Multidimensional cellular interrogation of the kidney in AKI and CKD

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Project Summary Current standard routine pathologic work-up of kidney biopsies has been in place for about 50 years and involves light microscopy (LM), immunofluorescent microscopy (IF)/immunohistochemistry (IHC), and electron microscopy (EM). Current practice of using kidney biopsies as one of the main diagnostic tools in the management of kidney has significant limitations. First, interpretation is founded on classic pathologic principles and approaches, which are by definition, descriptive, or semi-quantitative at most. Therefore, the interpretation has an inherent component of subjectivity and reproducibility and can be suboptimal which has been well documented both in the transplant and native kidney biopsy literature. Second, immunophenotypical characterization in the kidney is not part of the diagnostic work-up. Furthermore, in spite of advances in a number of various novel technologies potentially applicable to tissue interrogation of kidney biopsies, there have been no new technologies added to the diagnostic armamentarium with the sole exception of mass spectrometry for some limited applications. Therefore, it is evident that diagnostic potential of kidney biopsies is underutilized. This, coupled with the unmet need of better understanding the biology of acute kidney injury (AKI) and chronic kidney disease (CKD) makes kidney biopsy a potential high value source to acquire new knowledge through the application of novel tissue interrogation technologies. The ideal next generation tissue- based assay need to be highly reproducible, quantitative, fluorescence-based, and multiplexed to visualize multiple mRNAs and proteins at single cell resolution level. In this carefully designed study proposal we are going to serve as a Tissue Interrogation Site of the KPMP project. We will utilize two multiplex assays (1) multiplex Immunofluorescence and In Situ Hybridization (mIFISH) and (2) CODEX that have been developed by Dr. Nolan (Stanford) and by Dr. Laszik (UCSF) that fulfill the criteria listed above. The assays will use formalin-fixed paraffin-embedded (FFPE) tissue which has the best potential for standardization and to be representative due to its relatively large size. The new generation (in situ) assays that retain the structural integrity of tissues will be supplemented by additional ancillary tissue-based assays of proteomics and RNAseq on homogenized tissues which will be led by Drs. Sarwal and Sigdel at UCSF. Data generated by proteomics and RNAseq will be correlated with the conventional pathologic findings, and also with data generated on the in situ platforms of mIFISH and CODEX (i.e., tissue atlases). Cytometry by Time of Flight (CyTOF) assay will help interrogate the inflammatory compartment of AKI and CKD on fresh/cyoprotected samples. Data generated by proteomics, RNAseq, and CyTOF will also be used to identify bio-markers that will be incorporated into the in situ mIFISH and CODEX assays which are the “epicenter” of this proposal.