Detection of aging mechanisms of nephron degeneration using nanoscale pathology

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Summary With a rapidly growing elderly population in developed societies, and an organ shortage crisis caused by increasing transplantation demands, there is a pressing need to understand the biology of aging-associated organ deterioration. Loss of organ function throughout life encompasses progressive loss of parenchyma and accumulation of fibrosing connective tissue. In the aging kidney, nephron atrophy results in significant loss of tissue mass and overall decline in filtration capacity. Throughout the process, proximal tubule deterioration occurs concomitantly with excess extracellular matrix deposition in the basement membrane surrounding the tubules. The mechanisms that lead to tubulointerstitial degeneration with age remain largely unknown, this is due to the lack of understanding of the epithelial cell aging mechanisms and the molecular and structural changes associated with it. Because proximal tubule epithelial cell aging and progression of peritubular fibrosis are intimately related events, here we propose to study epithelial cell senescence and the impact of a senescence-associated secretory phenotype on the peritubular interstitium. Firstly, we propose to study aging- associated nuclear DNA events in PTECs from young, mature, middle aged and old mice to identify the initiation and progression of DNA damage into heterochromatin silencing complexes known as senescence- associated heterochromatin foci (SAHFs). Secondly, we will investigate changes in the peritubular interstitium in relation to the adoption of a profibrotic senescence-associated secretory phenotype by epithelial cells. To this end, we will study the behavior of peritubular fibroblasts and changes in extracellular matrix composition in relation to the formation of secretory vesicles containing connective tissue growth factor in senescing epithelial cells. To gain a novel insight into the structural chromatin changes as well as the extracellular matrix alterations, we will employ a novel microscopy technique termed Expansion Microscopy (ExM), which enables physical magnification of complex subcellular structures, allowing nanoscale resolution imaging of macromolecular complexes. The information generated in this pilot study will greatly contribute to our understanding of the mechanisms of aging-associated nephron senescence.