Regulated necrosis and failed repair in cisplatin-induced chronic kidney disease.

Murine Model of Cisplatin-Induced Chronic Kidney Disease The murine model of cisplatin-induced CKD was prepared as previously described with the following modification: kidneys were harvested at two weeks after the first dose and two weeks after the second dose of cisplatin. 10 Initially we selected a cisplatin dose of 20 mg/kg, however mice were unable to survive a second injection at this dose. The cisplatin dose of 15 mg/kg was ultimately chosen because animals would survive the first dose, which was almost completely resolved by the second dose, and could be repeated after two weeks, when chronic and irreversible injury was established and animals would survive in a state of chronic renal failure. Multiphoton Microscopy (MPM) Studies Tissue Clearing and Multiphoton Imaging and Processing Mouse kidney specimens were prepared, processed, and imaged using a tissue clearing protocol and multiphoton microscopy setup as previously described with minor modifications (see supplemental methods ). 10 Imaging was performed on a custom-built microscopy setup employing a Ti:Sapphire laser for excitation and a high numerical aperture and long working distance objective (Leica 0.95NA 1.95 mm WD) or with a shorter working distance objective with higher numerical aperture (Olympus 1.35NA) for higher resolution reconstructions. Second harmonic generation was simultaneously collected in transmittance during imaging. Tiled raster images for large reconstructions were collected overnight. Images were processed using standard ImageJ tools available with the FIJI distribution. Three-dimensional reconstructions were rendered by built-in volume viewing tools in FIJI.

Capsular Damage Analysis

Glomeruli were randomly marked on large volume 3D stacks taken at step sizes of 1-2um by employing the overlay function of ImageJ. Using customized scripts written in python, a cubical volume surrounding the position of marked glomeruli was extracted and composed into a randomized 3D mosaic containing glomeruli from all samples. The randomization ensured that the pathologist evaluating the level of capsular damage was blind to the time-point a given image originated from, reducing bias. Pathologists scored glomerular capsule damage based on reduction or visible damage to the cuboidal cells and categorized as 1) atubular, 2) clearly reduced/damaged, 3) normal, or 4) increased, indicated by score placed directly on glomerulus. The use of an 'increased' category reflects the finding that some glomerular capsules show higher percentage of cuboidal cell coverage than others, even in control specimens. Marked values were then linked with specimen of origin based on position of glomerulus on grid. Three to four kidneys resulting in at least 100 glomeruli/glomerular capsules at each time point were evaluated. Statistical comparisons of damage at different time points were done using the chi-square test for independence at a 0.01 level of significance. Imunohistochemistry (IHC) studies Immunohistochemical staining and scoring was performed for Ki67, F4/80, Tunel, α-smooth muscle actin (SMA), and CD34, as previously described, with blinded scoring performed by an expert renal pathologist using a square grid technique. 10 RNA and Protein Isolation RNA was isolated from mouse kidney using RNeasy ® Kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol. RNA concentrations were determined by absorbance at 260, 280, and 320 nm using a DS-11 Spectrophotometer (DeNovix Inc., Wilmington, DE). Kidney was lysed and homogenized in ice-cold RIPA buffer (Sigma, St. Louis, MO) supplemented with complete proteinase inhibitor (Roche Diagnostics, Risch-Rotkreuz, Switzerland) and phosphatase inhibitor (Sigma-Aldrich) cocktails using Bead Bug microtube homogenizer (Denville Scientific Inc., Holliston, MA) at 4,000rpm for 90 seconds. The tissue homogenates were centrifuged (13,000 × g , 15 min, 4 °C), supernatants were collected, and protein concentrations were determined (Bradford protein assay reagent, Bio-Rad.

Microarray Analysis

Microarray analysis was performed commercially with OneArray ® (Phalanx Biotech Group, San Diego, CA) with RNA samples isolated from control (N=3), 2 week-1 dose (N=3), and 4 week-2 dose mice(N=3). Qlucore Omics Explorer v 3.2 (Qlucore AB, Lund, Sweden) was used for identifying differentially-expressed genes (q < 0.01) using a one-way analysis of variance (ANOVA). Principal component analysis plots, unsupervised hierarchical clustering, and heat maps were also generated in Qlucore. Differentially expressed genes identified by Qlucore were subjected to further analysis by Ingenuity Pathway Analysis (Ingenuity Systems QIAGEN Build: 389077M, Content version: 27821452, Redwood City, CA, USA).

Western Blot Analysis

Equal amounts of protein lysate were separated by SDS-Page and western blotting was carried out using the following antibodies: NGAL, KIM1 (R&D Systems, Minneapolis, MN), phospho-JNK, phospho-ERK, total-ERK, RIPK1, and RIPK3 (Cell Signaling Technologies, Danvers, MA, USA). Proteins were detected using a chemiluminescent substrate (SuperSignal ® West Femto; Thermo Scientific, Rockford, IL, USA) and signal quantification was performed with ImageJ software (National Institutes of Health, Bethesda, MD, USA). More detailed methods are provided in the supplementary methods .

Supplementary Material 1