support for many collaborative studies, including antibody optimization and validation, pathologic interpretation, and semiquantitative and quantitative histopathologic analysis. Of direct relevance to this program project, Dr. Grande has worked closely with the Directors of Projects 1 and 2 in analyzing histologic sections obtained from the pig renal artery stenosis model (1-6). Dr. Grande has also collaborated with the Director of Project 4 in both clinical and experimental studies (4, 6-9).Dr. Grande has established an infrastructure for processing tissue sections, including a Dako Automated Stainer, which will minimize batch to batch variability in immunostaining protocols and a MetaVue Image analysis system which will facilitate quantitative analysis of markers of interstitial fibrosis, inflammation, and proliferation. Ms. Gina Warner has served as Lead Technician in Doctor Grande's laboratory for the past 8 years and is highly qualified to perform antibody development, tissue staining, and computer assisted quantitative image analysis. Ms. Warner will also be responsible for blinding the samples so they can be interpreted in an unbiased fashion. Sample Preparation. Renal tissue will be fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin, per standard techniques. Histologic sections, 4 jam, thick, will be prepared. Representative sections will be stained with H&E, PAS, and trichrpme stains. Additional unstained slides will be prepared for Sirius red staining, and immunohistochemical staining for a-smooth muscle actin (a-SMA), collagens III and IV, p-ERK, Mib-1, p21, p27, and TGF-p1. Immunostaining of renal tissue often cannot be performed by clinical immunopathology laboratories because the slides often have to be treated differently than other tissues. For example, high endogenous biotin content in renal tissue produces an extremely high background when protocols that work well in other tissues are employed. In addition, many of the antibodies that work on human tissues do not work well in murine or porcine tissue. The core will support the infrastructure necessary to systematically optimize and validate all of the antibodies which are an integral part of the experimental protocols outlined in Projects 1-4. Our laboratory has implemented a number of immunostains for clinical use and in support of research studies (see table for list of antibodies). Examples from studies published within the past two years are highlighted in references (6, 10-16) in the bibliography. Many of the antibodies (TGF-p1, matrix proteins, Mib-1, etc.) have already been validated in our research laboratory. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 327 Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): As necessary, antigen retrieval will be performed by heat treatment in EOTA for 30 minutes, using a vegetable steamer. Enzyme treatment will be performed as required for various antibodies (collagen IV requires pepsin treatment, TGF(31 and collagen III require trypsin treatment). Commercially available kits (Vectastain ABC kit, Vector Laboratories, and Envision Plus HRP kit, DakoCytomation) will be used for many of the blocking, secondary antibody and amplification steps. As necessary, reagents to block endogenous biotin activity will be used. Color development will be performed using NovaRed (Vector Laboratories) followed by hematoxylin counterstain. To facilitate consistency between staining batches, most of the stains will be performed on the Dako Autostainer, an automated staining machine. Staining reagents used with the machine include TBS-T, distilled water and DakoCytomation Special Stains Wash Buffer. Controls will include irrelevant isotype-specific antibody. A critical feature of the core is support of a dedicated technologist (Ms. Warner) who will standardize the staining protocols to ensure day-to-day reproducibility of the stains. Semiauantitative Histopatholoaic Analysis: Interpretation of studies outlined in Projects 1-4 is heavily dependent upon histopathologic analysis, which requires standardized and reproducible staining protocols (see above) as well as semiquantitative and quantitative histopathologic analysis, both of which will be provided by the Histopathology Core. The Core Director will perform semiquantitative histologic scoring, in a blinded fashion, on H&E, PAS, and trichrome stained slides; see Figure 1. The scoring system assigns points (0-3) for glomerular, interstitial, tubular, and vascular features of the renal tissue. The scoring system is similar to that previously used in a cohort of 148 biopsies obtained from patients with IgA nephropathy (17) and modified to include Banff criteria for assessment of renal biopsies (7, 18). In brief, glomerulosclerosis will be reported as number of sclerotic glomeruli, total glomeruli, and will be semiquantitatively analyzed as absent (0), mild (1, involving <25% of glomeruli), moderate (2, involving 26-50% of glomeruli), and severe (3, involving >50% of glomeruli). The number of segmentally sclerotic glomeruli will also be reported as a percent of total glomeruli and reported on a 0-3+ scale (see above). Additional glomerular features, including matrix increase, capillary loop narrowing, ischemic changes (wrinkling and folding), and mesangial cellularity will also be reported, as previously described. For the tubulointerstitial compartment, the extent of interstitial fibrosis, tubular atrophy, and interstitial infiltrates are scored as absent (0), involving <25% of the cortical surface area (1), involving 26-50% of the cortical surface area (2), and involving >50% of the cortical surface area (3). The presence of tubular dilatation, epithelial cell vacuolization, tubular atrophy, casts, or edema will be scored as absent (0), isolated (1), present in <10% of tubules (2), and present in >10% of tubules (3). Reduction in vascular caliber due to sclerosis or hyalinization is scored as absent (0), <25% luminal diameter PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 328 ,J. CoreD Figure 1A. Digital analysis of MT-stalned and oSMA- immunostained sections from renal allografts with newly-diagnosed CAN. MT-stained sections representative sections of renal cortex from biopsies with high interstitial fibrosis score (32.17%) (A, B) and low interstitial fibrosis score (12.64%) (C, D). Images are show before (A,C) and after (B,D) creation of a blue color-based threshold. oSMA immunoperoxidase-stained section - a representative section is shown before (E) and after (F) creation of a brown color-based threshold. Original magnification (10XFD). Figure 1B, Digital analysis of non-polarized and polarized Sirius Red-stained sections from renal allografts with newly-diagnosed CAN. A representative section is shown under non-polarized (A,B,C) and polarized (D,E,F) light. For each, the section is shown prior to (A,D) and after (B,E) conversion to a black and white Image and after thresholding (C,F) for black (non-polarized) or white (polarized) material. Original magnification (10XFD). Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): ROIflOrO, J. CaHOS COfG D (1), 26-75% diameter (2), and >75% (3). Intimal thickening is assessed by comparing the thickness of the intima relative to the total medial thickness and quantitated as normal (0), <25% medial thickness (1), intimal thickness <50% medial thickness (2), and intimal thickness >50% medial thickness (3). Medial hypertrophy is assessed by comparing the thickness of the muscular wall relative to the vascular caliber and is semiquantitatively assessed as normal (0), mild (1), moderate (2), or severe (3). Mean glomerular size and vascular media to total thickness ratios will be assessed by quantitative analysis (see below). Quantitative Assessment of Histopathologic Features, including Interstitial Fibrosis and Matrix Proteins: Methodologies will be as described in our recent publication (18); see Figure 1. Proliferative activity will be assessed as the number of positively staining nuclei/surface area of sections stained for Mib-1 (Projects 1-4), The MetaVue Image Analysis System will be used to evaluate mean glomerular planar surface area (of glomeruli containing the vascular pole) to complement the semiquantitative analysis of glomerular size described above. Glomerular size is used as a marker of renal hypertrophy. The core will assist with studies to determine protein/DNA ratios as an additional index of hypertrophy (DNA as assayed by spectrophotometric analysis of perchloric acid digest of renal cortex, as previously reported by us (19). Similarly, the number of cells staining positively for cell cycle regulatory molecules (p-ERK, p21, p27) will be assessed as the number of cells positively staining/cortical surface area. Glomeruli, tubules, interstitium, and vessels will be assessed separately. Interstitial fibrosis will be assessed over the entire renal cortex of Sirius red-stained slides as the relative amount of red staining material (observed under non-polarized light), or birefringent material (observed under polarized light), relative to the entire cortical surface area, as assessed by the MetaVue Image Analysis System. The percent of cortical surface area staining positively for a-smooth muscle actin will be quantitatively assessed with the MetaVue Image Analysis System. In serial sections, epithelial to mesenchymal transformation will be assessed as decreased E-cadherin staining of tubular epithelial cells, with increase in a- smooth muscle actin staining. This will be semiquantitatively reported as absent, mild, moderate, and severe (>50% cortical surface area). In select cases, we will perform double immunohistochemical staining for E- cadherin and a-SMA to identify and localize cells undergoing epithelial to mesenchymal transformation. Immunohistochemical stains for collagens III (as a marker of interstitial fibrosis) and IV (as a marker of tubular! basement membrane thickening), and TGF-p1 will be expressed as percent cortical surface area staining) positively for these markers, as assessed by the MetaVue Image Analysis System. TGF-B ELISA. We have developed this method for use in clinical samples and will support Project 4 (20, 21). Human urine samples (20 ml each, if possible) will be concentrated using Centriplus centrifugal filters with a 10,000 molecular weight cutoff (Amicon YM-10). Urines are centrifuged at 5000 x g at 25? C until the concentrate volume is approximately 300-500 j^l. Urines will be concentrated from 10- to 40-fold, depending on the start volume. Plasma samples do not require concentration. The ELISA will be performed using Quantikine TGF-pl ELISA Kits (R&D Systems, Minneapolis, MN). Kits are available for human samples. The assay will be completed following the R&D protocol, and the plate will be read on a Spectramax Plus 384 plate reader. Results will be in pg/mL after adjusting each sample for fold dilution and fold concentration. Statistical Analysis: All histologic sections will be read in a blinded fashion. Semiquantitative and quantitative data will be entered into an Excel Spreadsheet linked to the MetaVue Image Analysis System. The histotechnologist will calculate mean +/- SEM for all data. Comparisons between multiple groups (stenotic vs contralateral kidney; sham vs contralateral kidney) will be made by ANOVA, using Bonferroni correction for multiple comparisons, followed by the unpaired Students' t-test. D. Budget for Core D (See attached) PHS 398/2590 (Rev. 09/04, Reissued4/2006) Page 329 Continuation Format Page
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