Heme-mediated Mitochondrial Injury, Senescence, Acute Kidney Injury and Chronic Kidney Disease - PROJECT SUMMARY Senescent cells (SCs) cause senescence, a dominant risk factor for acute kidney injury (AKI). SCs are cell cycle-arrested (due to upregulated cell cycle inhibitors p16Ink4a and p21Cip1) and display a senescence- associated secretory phenotype (SASP) which is proinflammatory and proapoptotic. Senolytics, agents that kill SCs, are now in clinical trials. We demonstrate senescence in the heme protein-mediated AKI model (HP-AKI) as indicated by multiple indices. The significance of such changes – injurious or protective – is unknown as regards AKI. The AKI field recognizes that mitochondrial injury drives AKI, while the senescence field recognizes that mitochondrial injury elicits senescence; this application uniquely unites these two concepts. Early in HP-AKI, we demonstrate that mitochondria are injured and their heme content increased; normal mitochondria, exposed to such heme content, cease functioning. Heme, a prooxidant tetrapyrrole, present in the ubiquitous family of heme proteins, is freed when heme proteins are destabilized because of cellular stress. We also demonstrate that heme induces p16Ink4a/p21Cip1 and a SASP, and suppresses PGC-1α. In exploring heme-induced mitochondrial injury and induction of p16Ink4a/p21Cip1, we focused on two transcription factors both upregulated by mitochondrial injury, one, ETS1, being an inducer of p16Ink4a, the other, ATF4, an inducer of p21Cip1. Our preliminary data demonstrate that these 4 principal molecules (ETS1, ATF4, p16Ink4a, p21Cip1) are all induced in HP-AKI; in heme-exposed renal proximal tubular epithelial cells in vitro; and in renal ischemia-reperfusion injury (IRI). Our hypothesis is that AKI results from heme-mediated mitochondrial injury and ensuing senescence, a thesis to be tested in three aims. Aim I: Define the role of heme-mediated mitochondrial injury in ETS1 and ATF4 expression, senescence, and AKI. Using complementary in vivo and in vitro approaches, we will sequentially examine the role of heme-mediated mitochondrial injury; the contribution of ETS1, ATF4, and ETS1/ATF4-independent pathways; and the involvement of senescence in AKI. Aim II: Define the roles of p21Cip1 and p16Ink4a in AKI: Genetic strategies. The role of p21Cip1 in AKI will be examined by inducible deletion of high p21Cip1-expressing cells and with proximal tubule-specific p21 KO mice. The role of p16Ink4a will be examined by inducible deletion of high p16Ink4a-expressing cells and by inducible proximal tubule-specific deletion of high p16Ink4a-expressing cells. Aim III: Define the effect of senolytics in AKI. SCs survive because of upregulated anti-apoptotic pathways, while senolytics kill SCs but not non-SCs. This aim examines the efficacy of senolytics in AKI, and in reducing the sensitivity of aged mice to AKI. In sum, this resubmitted R01 examines senescence in AKI, linking it sequentially to heme-mediated mitochondrial injury, the transcription factors ETS1 and ATF4, and p16Ink4a and p21Cip1. This R01 offers novel insights regarding the role of senescence in the pathogenesis of AKI and the therapeutic utility of senolytics.
