Insights into protein-bound uremic toxins in proximal tubule cell senescence and kidney fibrosis

Abstract

Kidney fibrosis leads to kidney failure by an excessive accumulation of extracellular matrix (ECM), which is the common endpoint for a variety of progressive chronic kidney diseases (CKD). In healthy kidneys, protein-bound uremic toxins (PBUTs) are cleared from the systemic circulation by proximal tubule cells through the concerted action of plasma membrane transporters that facilitate their urinary excretion, but the endogenous metabolites are hardly removed with kidney dysfunction. Accumulating evidence suggests that senescence of kidney tubule cells influences kidney fibrosis. Senescence is a special state of cells characterized by permanent cell cycle arrest and limitation of proliferation, which promotes fibrosis by releasing senescence-associated secretory phenotype (SASP) factors. This thesis demonstrated the effects related to senescence of PBUTs in vitro by using conditionally immortalized proximal tubule epithelial cell line overexpressing the organic anion transporter 1 (ciPTEC-OAT1), which gives a new angle to the role that PBUTs play in CKD. These observations thus pave the way for investigating novel strategies for CKD treatment, such as kidney-targeted delivery of senolytic drugs. Chapter 1 gives a general introduction of kidney fibrosis, senescence and PBUTs. A hypothesis is proposed that PBUTs may promote kidney fibrosis by accelerating senescence, possibly via mitochondrial dysfunction, cell cycle arrest, and the production of SASP factors. The findings described in Chapter 2 suggest that ciPTEC-OAT1 develops a senescence phenotype in a time dependent manner at 37℃, including a cell cycle arrest, resistance to apoptosis, SASP factors production and responsiveness to senolytics treatment. Therefore, ciPTEC-OAT1 represents a valid model for studying kidney senescence by simply adjusting culture conditions. Chapter 3 and Chapter 4 indicate that PBUTs could induce SASP factors release, and trigger oxidative stress possibly causing mitochondrial dysfunction, thus driving kidney senescence. The results in Chapter 3 demonstrate that PBUTs induce inflammasome-mediated IL-1β production in proximal tubule cells via oxidative stress and NF-κB signalling. Chapter 4 indicates that IS may contribute to kidney disease by accelerating senescence, through the regulation of senescence markers and by modulating inflammatory and profibrotic processes, as evidenced by changes in the TNF-α/NF-ĸB pathway and the EMT process. Together, the findings revealed that PBUTs, and especially IS, are drivers of kidney senescence, and that they do so by inducing oxidative stress, promoting inflammatory response, and via increased resistance to cell death. Chapter 5 provides insight into possible approaches for kidney-targeted senolytic therapy (Nav-Lx-LZM conjugates) for more efficient clearance of senescent cells and the reduction of systemic side effects. The results obtained suggest that ciPTEC-OAT1 appears suitable to take up the rhodamine-LZM conjugates, indicating that this is a valuable in vitro model to evaluate the efficacy of senolytic-LZM conjugates. Finally, Chapter 6 gives an overview on how senescence contributes to CKD and the involvement of PBUTs, how kidney senescence can be modelled and studied, the ways of improving current senolytic therapies, along with future perspectives.