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 PointsUnderstanding 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.BackgroundUnderstanding 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.MethodsNAD+-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.ResultsThe 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.ConclusionsThese 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.

Angiotensin-converting enzyme 2 (ACE2) exerts countervailing effects on the renin-angiotensin-aldosterone system (RAAS). ACE2 also engages the spike protein of SARS-CoV-2. ACE2 protein has been shown recently to avidly bind heme. We examined the pathobiological relevance of this heme-binding property of ACE2 by using the glycerol-induced model of heme protein-mediated AKI (HP-AKI), which is characterized by increased kidney heme content. We studied the response of ACE2-wildtype (ACE2+/y) and ACE2-deficient (ACE2-/y) mice to HP-AKI and quantitated kidney and cellular content of heme under relevant conditions. ACE2-deficient mice, compared with ACE2-wildtype mice, were significantly protected against HP-AKI as reflected by filtration markers, less histological injury, and less expression of apoptosis and ferroptosis markers. ACE2-deficient mice also evinced lesser kidney heme content and a blunted induction of HO-1. HEK293 ACE2-overexpressing cells, compared with HEK293-native cells, when exposed to heme, retained higher amounts of heme. In HP-AKI, ACE2 expression and activity were reduced, and myoglobin and heme, administered independently, reduced ACE2 expression in the otherwise intact mouse kidney. Finally, with more severe HP-AKI, the protective effect of ACE2 deficiency was attenuated. We conclude that ACE2 deficiency confers protection against HP-AKI. We suggest that this reflects the recently recognized binding of heme to ACE2, such binding serving to facilitate renal entry of heme, a known nephrotoxin. These findings uncover a novel pathway of heme-dependent acute kidney injury. This is the first demonstration of the biological relevance of chemical binding of heme by ACE2. Finally, we identify heme proteins and heme as novel determinants of ACE2 expression.NEW & NOTEWORTHY 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 PointsThe rat arteriovenous fistula (AVF) model exhibits marked upregulation of p16Ink4a and p21Cip1 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.BackgroundMaturational 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.MethodsThe 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 p16Ink4a and p21Cip1, and such indices of a senescence phenotype as senescence-associated beta-galactosidase (SA-beta-gal) activity, SA-beta-gal staining, and a senescence-associated secretory phenotype. We examined the effects of compounds that retard or accelerate senescence on AVF blood flow.ResultsThe AVF evinced upregulation of p16Ink4a and p21Cip1 when assessed 3 days after AVF creation. The AVF also demonstrated increased SA-beta-gal activity in the artery and vein; staining for SA-beta-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.ConclusionsThe 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.

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, p16Ink4a, 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 p16Ink4a expression, murine models of genetic deficiency of hemopexin (HPX) and heme oxygenase-1 (HO-1) were used. To determine the involvement of p16Ink4a in HP-AKI, the population of p16Ink4a-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 p16Ink4a. p16Ink4a 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 p16Ink4a 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 p16Ink4a-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 p16Ink4a, the latter driven, in part, by hemoglobin, myoglobin, and heme.

Croatt, Anthony J.; Grande, Joseph P.; Ackerman, Allan W.; Singh, Raman D.; Nath, Karl A. Author Information

Background Mitochondrial injury occurs in and underlies acute kidney injury (AKI) caused by ischemiareperfusion and other forms of renal injury. However, to date, a comprehensive analysis of this issue has not been undertaken in heme protein-induced AKI (HP-AKI). We examined key aspects of mitochondrial function, expression of proteins relevant to mitochondrial quality control, and mitochondrial ultrastructure in HP-AKI, along with responses to heme in renal proximal tubule epithelial cells.Methods The long-established murine glycerol model of HP-AKI was examined at 8 and 24 hours after HP-AKI. Indices of mitochondrial function (ATP and NAD(+)), expression of proteins relevant to mitochondrial dynamics, mitochondrial ultrastructure, and relevant gene/protein expression in heme-exposed renal proximal tubule epithelial cells in vitro were examined.Results ATP and NAD(+) content and the NAD+/NADH ratio were all reduced in HP-AKI. Expression of relevant proteins indicate that mitochondrial biogenesis (PGC-1a, NRF1, and TFAM) and fusion (MFN2) were impaired, as was expression of key proteins involved in the integrity of outer and inner mitochondrial membranes (VDAC, Tom20, and Tim23). Conversely, marked upregulation of proteins involved in mitochondrial fission (DRP1) occurred. Ultrastructural studies, including novel 3D imaging, indicate profound changes in mitochondrial structure, including mitochondrial fragmentation, mitochondrial swelling, and misshapen mitochondrial cristae; mitophagy was also observed. Exposure of renal proximal tubule epithelial cells to heme in vitro recapitulated suppression of PGC-1a (mitochondrial biogenesis) and upregulation of p-DRP1 (mitochondrial fission).Conclusions Modern concepts pertaining to AKI apply to HP-AKI. This study validates the investigation of novel, clinically relevant therapies such as NAD(+)-boosting agents and mitoprotective agents in HP-AKI.

Heme proteins, the stuff of life, represent an ingenious biologic strategy that capitalizes on the biochemical versatility of heme, and yet is one that avoids the inherent risks to cellular vitality posed by unfettered and promiscuously reactive heme. Heme proteins, however, may be a double-edged sword because they can damage the kidney in certain settings. Although such injury is often viewed mainly within the context of rhabdomyolysis and the nephrotoxicity of myoglobin, an increasing literature now attests to the fact that involvement of heme proteins in renal injury ranges well beyond the confines of this single disease (and its analog, hemolysis); indeed, through the release of the defining heme motif, destabilization of intracellular heme proteins may be a common pathway for acute kidney injury, in general, and irrespective of the underlying insult. This brief review outlines current understanding regarding processes underlying such heme protein-induced acute kidney injury (AKI) and chronic kidney disease (CKD). Topics covered include, among others, the basis for renal injury after the exposure of the kidney to and its incorporation of myoglobin and hemoglobin; auto-oxidation of myoglobin and hemoglobin; destabilization of heme proteins and the release of heme; heme/iron/oxidant pathways of renal injury; generation of reactive oxygen species and reactive nitrogen species by NOX, iNOS, and myeloperoxidase; and the role of circulating cell-free hemoglobin in AKI and CKD. Also covered are the characteristics of the kidney that render this organ uniquely vulnerable to injury after myolysis and hemolysis, and pathobiologic effects emanating from free, labile heme. Mechanisms that defend against the toxicity of heme proteins are discussed, and the review concludes by outlining the therapeutic strategies that have arisen from current understanding of mechanisms of renal injury caused by heme proteins and how such mechanisms may be interrupted.

Discovering new nephroprotectants may provide therapeutic strategies in AKI.This study provides the first evidence that KLF11, a member of the Krüppel-like factor (KLF) family of proteins, protects against AKI.In the absence of KLF11, exaggerated induction of endothelin-1 and IL-6 occurs after ischemic renal injury and may contribute to worse AKI.

Background The actions of angiotensin-converting enzyme 2 (ACE2) oppose those of the renin-angiotensin-aldosterone system. ACE2 may be a cytoprotectant in some tissues. This study examined ACE2 expression in models of AKI. Methods ACE2 mRNA and protein expression and ACE2 activity were assessed in murine ischemic AKI. Renal ACE2 mRNA expression was evaluated in LPS-induced AKI in wild-type (C57BL/6J) mice, in heme oxygenase-1+/+ and heme oxygenase-1 / mice, and after unilateral ureteral obstruction (UUO) in wild-type mice. The effect of sex and age on renal ACE2 protein expression was also assessed. Results In ischemic AKI, ACE2 mRNA and protein expression and ACE2 activity were reduced as compared with such indices in the intact kidney. In ischemic AKI, ACE2, which, in health, is prominently expressed in the tubular epithelium, especially proximal tubules, is decreased in expression in these segments. Decreased ACE2 expression in AKI did not reflect reduced GFR, because ACE2 mRNA expression was unaltered after UUO. LPS induced renal ACE2 mRNA expression in wild-type mice, but this effect did not occur in heme oxygenase-1-deficient mice. In ischemic and LPS-induced AKI, renal expression of the Mas receptor was increased. In the intact kidney, renal ACE2 protein expression decreased in female mice as compared with male mice, but was unaltered with age. Conclusion We conclude that renal ACE2 expression is decreased in ischemic AKI, characterized by decreased GFR and abundant cell death, but is upregulated in LPS-induced AKI, an effect requiring heme oxygenase-1. Determining the significance of ACE2 expression in experimental AKI merits further study. We suggest that understanding the mechanism underlying ACE2 downregulation in AKI may offer insights relevant to COVID-19: ACE2 expression is downregulated after ACE2 mediates SARS-CoV-2 cellular entry; such downregulation is proinflammatory; and AKI commonly occurs and determines outcomes in COVID-19.

BACKGROUND:Acute kidney injury (AKI) is both a consequence and determinant of outcomes in COVID-19. The kidney is one of the major organs infected by the causative virus, SARS-CoV-2. Viral entry into cells requires the viral spike protein, and both the virus and its spike protein appear in the urine of COVID-19 patients with AKI. We examined the effects of transfecting the viral spike protein of SARS-CoV-2 in kidney cell lines. METHODS:HEK293, HEK293-ACE2+ (stably overexpressing ACE2), and Vero E6 cells having endogenous ACE2 were transfected with SARS-CoV-2 spike or control plasmid. Assessment of gene and protein expression, and syncytia formation was performed, and the effects of quercetin on syncytia formation examined. FINDINGS:Spike transfection in HEK293-ACE2+ cells caused syncytia formation, cellular sloughing, and focal denudation of the cell monolayer; transfection in Vero E6 cells also caused syncytia formation. Spike expression upregulated potentially nephrotoxic genes (TNF-α, MCP-1, and ICAM1). Spike upregulated the cytoprotective gene HO-1 and relevant signaling pathways (p-Akt, p-STAT3, and p-p38). Quercetin, an HO-1 inducer, reduced syncytia formation and spike protein expression. INTERPRETATION:The major conclusions of the study are: 1) Spike protein expression in kidney cells provides a relevant model for the study of maladaptive and adaptive responses germane to AKI in COVID-19; 2) such spike protein expression upregulates HO-1; and 3) quercetin, an HO-1 inducer, may provide a clinically relevant/feasible protective strategy in AKI occurring in the setting of COVID-19. FUNDING:R01-DK119167 (KAN), R01-AI100911 (JPG), P30-DK079337; R01-DK059600 (AA).

Background: Acute kidney injury (AKI) is both a consequence and determinant of outcomes in COVID-19. The kidney is one of the major organs infected by the causative virus SARS-CoV-2. The spike protein of SARS-CoV-2 is required for viral entry into cells and is present in the urine of patients with COVID-19 and AKI. The present study examined cellular effects that result from transfecting the spike protein of SARS-CoV-2 in HEK293 kidney cells. Methods: HEK293-ACE2+ cells stably overexpressing ACE2 were used. Codon optimized pcDNA encoding SARS-CoV-2 spike (7788bp) or empty vector (4033bp) plasmid was transfected using Lipofectamine LTX. For studies examining the effect of quercetin (an inducer of heme oxygenase-1, HO-1), full media containing quercetin or vehicle was added at 4-6 hours post transfection. mRNA and protein expression was assessed by quantitative real-time RT-PCR and western blot respectively. Syncytium formation was assessed by acquiring phase contrast images using Olympus CK40 microscope and the area covered by syncytia was measured using ImageJ software. Results: HEK293-ACE2+ cells expressed SARS-CoV-2 spike protein upon spike transfection. Such expression led to syncytia formation, the sloughing of sheets of cells, and focal denudation of the cell monolayer. Spike protein expression upregulated potentially nephrotoxic genes such as TNF-α, MCP-1, and ICAM1. Spike protein expression also upregulated potentially cytoprotective genes such as HO-1, as demonstrated by HO-1 mRNA and protein expression and relevant signaling pathways (p-Akt, p-STAT3, and p-p38) involved in inducing the HO-1 gene. Quercetin, a naturally occurring compound that induces HO-1, markedly reduced syncytia formation and spike protein expression. Conclusions: These findings introduce a clinically relevant, spike protein-induced, in vitro model for the study of AKI in COVID-19. The major conclusions of the study are: 1) Spike protein expression in kidney cells provides a useful and timely model for the study of maladaptive and adaptive responses in these cells relevant to AKI observed in COVID-19;2) spike protein expression in kidney cells upregulates HO-1;and 3) quercetin, an inducer of HO-1, may provide a clinically relevant/feasible protective strategy in AKI occurring in the setting of COVID-19.

Restoration of kidney tubular epithelium following sublethal injury sequentially involves partial epithelial-mesenchymal transition (pEMT), proliferation, and further redifferentiation into specialized tubule epithelial cells (TECs). Because the immunosuppressant cyclosporine-A produces pEMT in TECs and inhibits the peptidyl-prolyl isomerase (PPIase) activity of cyclophilin (Cyp) proteins, we hypothesized that cyclophilins could regulate TEC phenotype. Here we demonstrate that in cultured TECs, CypA silencing triggers loss of epithelial features and enhances transforming growth factor β (TGFβ)-induced EMT in association with upregulation of epithelial repressors Slug and Snail. This pro-epithelial action of CypA relies on its PPIase activity. By contrast, CypB emerges as an epithelial repressor, because CypB silencing promotes epithelial differentiation, prevents TGFβ-induced EMT, and induces tubular structures in 3D cultures. In addition, in the kidneys of CypB knockout mice subjected to unilateral ureteral obstruction, inflammatory and pro-fibrotic events were attenuated. CypB silencing/knockout leads to Slug, but not Snail, downregulation. CypB support of Slug expression depends on its endoplasmic reticulum location, where it interacts with calreticulin, a calcium-buffering chaperone related to Slug expression. As CypB silencing reduces ionomycin-induced calcium release and Slug upregulation, we suggest that Slug expression may rely on CypB modulation of calreticulin-dependent calcium signaling. In conclusion, this work uncovers new roles for CypA and CypB in modulating TEC plasticity and identifies CypB as a druggable target potentially relevant in promoting kidney repair.

In the murine venous thrombosis model induced by ligation of the inferior vena cava (IVCL), genetic deficiency of heme oxygenase-1 (HO-1) increases clot size. This study examined whether induction of HO-1 or administration of its products reduces thrombosis. Venous HO-1 upregulation by gene delivery reduced clot size, as did products of HO activity, biliverdin, and carbon monoxide. Induction of HO-1 by hemin reduced clot formation, clot size, and upregulation of plasminogen activator inhibitor-1 (PAI-1) that occurs in the IVCL model, while leaving urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) expression unaltered. The reductive effect of hemin on clot size required HO activity. The IVCL model exhibited relatively high concentrations of heme that peaked just before maximum clot size, then declined as clot size decreased. Administration of hemin decreased heme concentration in the IVCL model. HO-2 mRNA was induced twofold in the IVCL model (vs. 40-fold HO-1 induction), but clot size was not increased in HO-2−/− mice compared with HO-2+/+ mice. Hemopexin, the major heme-binding protein, was induced in the IVCL model, and clot size was increased in hemopexin−/− mice compared with hemopexin+/+ mice. We conclude that in the IVCL model, the heme-degrading protein HO-1 and HO products inhibit thrombus formation, as does the heme-binding protein, hemopexin. The reductive effects of hemin administration require HO activity and are mediated, in part, by reducing PAI-1 upregulation in the IVCL model. We speculate that HO-1, HO, and hemopexin reduce clot size by restraining the increase in clot concentration of heme (now recognized as a procoagulant) that otherwise occurs. NEW & NOTEWORTHY This study provides conclusive evidence that two proteins, one heme-degrading and the other heme-binding, inhibit clot formation. This may serve as a new therapeutic strategy in preventing and treating venous thromboembolic disease.