Nath, Karl A.

Aging is unavoidable and driven, at least in part, by the biologic process termed senescence. Senescence is characterized by cell cycle arrest incurred by one or more stressors wherein there is cellular upregulation of cyclin-dependent kinase inhibitors, p16 Ink4a and/or p21 Cip1 . Senescent cells are metabolically active and resistant to apoptosis, have increased β -galactosidase activity, and express a senescence-associated secretory phenotype (SASP). The SASP produces an array of inflammatory, cytotoxic, fibrotic, and vasoactive factors. Senescence is a diffusible process as, largely through the SASP, a focus of senescent cells can recruit adjacent cells and cells in distant organs, thereby expanding its scope way beyond its original focus. Senescence predisposes not only to chronic diseases (including CKD) but also acute processes (including AKI). This review provides evidence that age increases the risk of human AKI and summarizes clinical factors that predispose older individuals to AKI. It provides an overview of the biology of senescence, and it discusses the role of intrarenal senescent cells in the pathogenesis of AKI, the significance of the extrarenal milieu and distant organs in predisposing to AKI with age, heme as a prosenescent species, and the role of senescence in the AKI to CKD transition. The risk for AKI with aging, in large part, reflects the fact that virtually all processes implicated in AKI, irrespective of age, occur as the kidney ages. Processes discussed include senescent cells, vascular responses, diabetes, impaired NAD + content, sirtuin expression, mitochondrial dysfunction, impaired autophagy, epigenetic changes, telomere shortening, impaired nephroprotectant expression, inflammaging, and immunosenescence. The review concludes by discussing the basis for senolytics (agents that kill senescent cells), senomorphics (agents that block SASP factors), and other aspects of senotherapies.

Key Points Understanding how heme proteins/heme induce AKI and whether NAD + -boosting ameliorates heme protein–mediated AKI is clinically relevant. Heme proteins/heme directly decrease kidney NAD + content and NAD + -boosting is markedly protective in heme protein–mediated AKI. These findings provide new insights in heme protein–mediated AKI and support interest in NAD + -boosting as a therapeutic strategy in AKI. Background Understanding how heme proteins and heme induce AKI is clinically relevant from numerous perspectives, and, in this regard, a widely used model of heme protein–mediated AKI (HP-AKI) involves the glycerol model in mice. Using this model, we have previously demonstrated that NAD + content is decreased in HP-AKI. Because there is significant current interest in NAD + -boosting as a strategy in clinical AKI, we examined the effect of NAD + -boosting in this model. Methods NAD + -boosting was achieved by the administration of nicotinamide mononucleotide (NMN) in the glycerol model of HP-AKI. The effect of NMN, compared with vehicle, was examined in mice with HP-AKI and with sham AKI at day 1 after HP-AKI was induced. Results The administration of NMN preserved kidney NAD + content in HP-AKI, and compared with the administration of vehicle in HP-AKI, NMN improved glomerular filtration markers; reduced histologic injury as assessed by tubular necrosis, dilation, and cast formation and by assessment of dystrophic calcification; reduced expression of kidney injury marker 1, a sensitive marker of AKI; preserved mitochondrial ultrastructure and increased expression of proteins that promote mitochondrial integrity; reduced apoptosis as assessed by terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling staining and genes and protein expression that contribute to apoptosis, while increasing expression of an anti-apoptotic gene; reduced expression of several renal injury-related genes; and mitigated the severity of the senescence phenotype, as assessed by multiple markers. Finally, the administration of myoglobin or heme in vivo diminished kidney NAD + content in mice with intact kidneys. Conclusions These findings demonstrate the remarkable protective effects of NAD + -boosting by NMN in HP-AKI as revealed by a multitude of markers relevant to AKI. The reduction in NAD + content in the kidney in HP-AKI may reflect the direct effects of heme proteins and/or heme. We suggest that these findings support current interest in NAD + -boosting as a therapeutic strategy in clinical AKI.

Background We aimed to assess the longitudinal changes of glycocalyx, a proteoglycan/glycoprotein layer that lines and protects endothelial cells in normotensive pregnancies (NPs); to correlate glycocalyx parameters with kidney injury markers (podocin‐positive to nephrin‐positive [P/N] ratio); and to explore changes in P/N ratio in preeclampsia. Methods Plasma and urine samples and glycocalyx measurements using side‐stream darkfield imaging were collected at each trimester from pregnant women (n=31). Plasma glycocalyx components were measured by ELISA, and urinary P/N‐positive extracellular vesicles by flow cytometry. Results In NPs (n=26), glycocalyx degradation increased from the first to the second trimester ( P =0.008) and returned to first‐trimester values in the third trimester ( P =0.008). Microvascular perfusion decreased from the first to the second trimester ( P =0.006) and increased back to first‐trimester values by the third trimester ( P =0.039). Plasma syndecan 1 level showed a steady increase from the first to the second trimester ( P <0.001) and further in the third trimester ( P <0.001); hyaluronic acid increased from the first to the second trimester ( P =0.016) without further changes. Glycocalyx degradation was associated with an increase in P/N ratio from the first to the second trimester of NPs and remained stable through the third trimester. P/N ratio strongly correlated with elevated syndecan 1 and hyaluronic acid levels in the third trimester. Comparison of P/N ratios between NP and preeclampsia (n=5) showed higher values in preeclampsia across all trimesters ( P =0.028). Conclusions Glycocalyx changes during NPs are associated with elevated urinary podocyte markers. Increase in P/N ratio across NPs, and further in preeclampsia, suggests that podocyte injury in preeclampsia is extension of physiological changes in pregnancy, which may predate its clinical presentation.

ACE2 protein binds heme, which we reasoned would promote heme entry into the kidney and, accordingly, heme protein-mediated acute kidney injury. Our findings support this hypothesis. This study is the first to demonstrate the biological relevance of ACE2-heme binding, uncover a new pathway of heme-dependent kidney injury, and identify myoglobin and heme as novel determinants of ACE2 expression. Our study explains why plasma levels of myoglobin and heme predict poor outcomes in patients with COVID-19.

Key Points The rat arteriovenous fistula (AVF) model exhibits marked upregulation of p16 Ink4a and p21 Cip1 and multiple markers of senescence. Fisetin, an established vasoprotective senolytic agent, when administered for 3 weeks, increases AVF blood flow and AVF outward remodeling. Heme is shown to be a novel prosenescence metabolite, and when chronically administered, it decreases AVF blood flow. Background Maturational failure of dialysis arteriovenous fistulas (AVFs) not uncommonly occurs and is of considerable and timely importance. Our prior studies demonstrate that senescence, a phenotypic process that promotes vascular and other diseases, occurs in the murine AVF. In this study, we examined whether senescence also occurs in the rat AVF model and the effect of compounds that inhibit or accelerate senescence. Methods The rat AVF was created in the femoral vessels by an end vein-side artery anastomosis. In the AVF, we assessed the expression of critical drivers of senescence, specifically, the cell cycle inhibitors p16 Ink4a and p21 Cip1 , and such indices of a senescence phenotype as senescence-associated β -galactosidase (SA- β -gal) activity, SA- β -gal staining, and a senescence-associated secretory phenotype. We examined the effects of compounds that retard or accelerate senescence on AVF blood flow. Results The AVF evinced upregulation of p16 Ink4a and p21 Cip1 when assessed 3 days after AVF creation. The AVF also demonstrated increased SA- β -gal activity in the artery and vein; staining for SA- β -gal in the AVF artery, anastomosis, and vein; and a prominent senescence-associated secretory phenotype. Fisetin, an established senolytic that is protective in other models of vascular injury, when administered for 3 weeks, increased AVF blood flow and outward remodeling. Hemin, when administered for 3 weeks, decreased AVF blood flow. We demonstrate that hemin is a novel inducer of a senescence phenotype in endothelial cells, as reflected by several senescence indices. However, when administered relatively acutely (for 5 days), hemin increased AVF blood flow by heme oxygenase–dependent mechanisms because the latter was entirely prevented by a competitive inhibitor of heme oxygenase activity. Conclusions The rat AVF exhibits senescence within 3 days of its creation. Chronic administration of a senolytic compound (fisetin) increases AVF blood flow, whereas chronic administration of a prosenescence compound (hemin) decreases AVF blood flow.

Key Points In heme protein–mediated AKI (HP-AKI), a senescence phenotype promptly occurs, and increased expression of p16 Ink4a contributes to HP-AKI. Renal p16 Ink4a expression is induced by hemoglobin, myoglobin, and heme in vivo and in renal epithelial cells exposed to heme in vitro . Impairing the binding or degradation of heme by hemopexin deficiency or heme oxygenase-1 deficiency, respectively, further upregulates p16 Ink4a . Background Understanding the pathogenetic basis for AKI involves the study of ischemic and nephrotoxic models of AKI, the latter including heme protein–mediated AKI (HP-AKI). Recently, interest has grown regarding the role of senescence as a mechanism of kidney injury, including AKI. We examined whether senescence occurs in HP-AKI and potential inducers of and the role of a key driver of senescence, namely, p16 Ink4a , in HP-AKI. Methods The long-established murine glycerol model of HP-AKI was used, and indices of senescence were examined. To evaluate the interaction of heme and p16 Ink4a expression, murine models of genetic deficiency of hemopexin ( HPX ) and heme oxygenase-1 ( HO-1 ) were used. To determine the involvement of p16 Ink4a in HP-AKI, the population of p16 Ink4a -expressing cells was reduced using the INK-ATTAC model. Results Using multiple indices, a senescence phenotype appears in the kidney within hours after the induction of HP-AKI. This phenotype includes significant upregulation of p16 Ink4a . p16 Ink4a is upregulated in the kidney after the individual administration of myoglobin, hemoglobin, and heme, as well as in renal epithelial cells exposed to heme in vitro . Genetic deficiencies of HPX and HO-1 , which, independently, are expected to increase heme content in the kidney, exaggerate induction of p16 Ink4a in the kidney and exacerbate HP-AKI, the latter shown in the present studies involving HPX −/− mice and in previous studies involving HO-1 −/− mice. Finally, reduction in the population of p16 Ink4a -expressing cells in the kidney improves renal function in HP-AKI even within 24 hours. Conclusions The pathogenesis of HP-AKI involves senescence and the induction of p16 Ink4a , the latter driven, in part, by hemoglobin, myoglobin, and heme.