Multiplexed Imaging of Senescent Chromatin State in Single Cells in the Kidneys

Background: The gradual loss of kidney function with age and disease can be linked to changes in physiology and single cell epigenetics. As cells become stressed or damaged from these conditions, they undergo the process of becoming senescent, a state of permanent cell cycle arrest associated with massive chromatin rearrangement and telomere shortening, as a way to mitigate further damage. The order of events and extent of epigenetic changes within single cells and their correlation to physiological alterations as the kidney ages or becomes diseased and senescence forms are not fully understood. Methods: Super-resolution optical microscopy techniques were used with advanced chemical labeling methods to concurrently study epigenetic states and nanoscale physiology in mouse kidney slices. We used multiplexed imaging to simultaneously study histone marks, telomeres, and tissue morphology at the single cell level. Results: The use of optical super-resolution techniques allows for ~70 nm spatial resolution. Multiple histone modifications have been detected and quantified throughout the nucleus and at locations of repetitive DNA sequences (telomeres and major satellites) in single cells with a high degree of accuracy. In the same sample, these single cell epigenetic signatures were correlated with nanoscale features within glomeruli and proximal tubules. Telomeres in aging samples have been shown to shorten using imaging techniques. The quantification of these nanoscale features includes the glomerular basement membrane thickness, width of individual podocyte foot processes, and the size of endothelial fenestrations. Conclusion: The combination of these advanced labeling methods and super-resolution optical microscopy techniques allows for an unprecedented view and correlation of single cell epigenetic states and nanoscale physiology in kidney tissue at many age points. Our approach has implications for identifying specific epigenetic changes in the kidney that precede the development of senescence and related diseases and their physiological markers. Understanding the sequence of events may assist in predicting disease formation along with studying the effectiveness of various treatments. Funding: NIDDK Support