Targeting Senescent Cells as Therapy for CKD.

Senescent cells accumulate in the kidney with aging, after acute and chronic injuries, and are present in increased numbers in deteriorating kidney transplants. Senescent cells have undergone permanent cell cycle arrest and release many proinflammatory cytokines/chemokines and profibrotic factors: the senescence-associated secretory phenotype. Recent work from several groups including our own has shown that senescent cells play a causative role in progression of kidney disease. Experimental evidence also indicates that targeting senescent cells has potential to alter the renal regenerative response, reducing progressive fibrosis and improving functional recovery after injury. Research and clinical interest is focused on understanding how accumulating chronic senescent cells link acute injury to progressive fibrosis, dysfunction, and mortality in human CKD. In this review, we outline current protocols for the identification of how senescent cells are identified in vitro and in vivo . We discuss the proposed mechanisms of actions of first-generation senolytic and senomorphic agents, such as ABT-263 (navitoclax) which targets the BCL2 family of survival factors, and senomorphic agents such as metformin which targets aspects of the senescence-associated secretory phenotype. We also review that emerging technologies, such as nanocarriers, are now being developed to have safer delivery systems for senolytics, greater specificity, fewer off-target effects, and less toxicity. Other methods of senescent cell elimination being developed target various immune evasion tactics displayed by these cells. By understanding the role of senescence in kidney homeostasis and disease, developing new, targeted compounds and the tools to allow their efficacy to be charted noninvasively, it should become possible for senolytic treatments to move from the bench to bedside.

Indian Hedgehog release from TNF-activated renal epithelia drives local and remote organ fibrosis.

Progressive fibrosis is a feature of aging and chronic tissue injury in multiple organs, including the kidney and heart. Glioma-associated oncogene 1 expressing (Gli1+) cells are a major source of activated fibroblasts in multiple organs, but the links between injury, inflammation, and Gli1+ cell expansion and tissue fibrosis remain incompletely understood. We demonstrated that leukocyte-derived tumor necrosis factor (TNF) promoted Gli1+ cell proliferation and cardiorenal fibrosis through induction and release of Indian Hedgehog (IHH) from renal epithelial cells. Using single-cell-resolution transcriptomic analysis, we identified an "inflammatory" proximal tubular epithelial (iPT) population contributing to TNF- and nuclear factor κB (NF-κB)-induced IHH production in vivo. TNF-induced Ubiquitin D (Ubd) expression was observed in human proximal tubular cells in vitro and during murine and human renal disease and aging. Studies using pharmacological and conditional genetic ablation of TNF-induced IHH signaling revealed that IHH activated canonical Hedgehog signaling in Gli1+ cells, which led to their activation, proliferation, and fibrosis within the injured and aging kidney and heart. These changes were inhibited in mice by Ihh deletion in Pax8-expressing cells or by pharmacological blockade of TNF, NF-κB, or Gli1 signaling. Increased amounts of circulating IHH were associated with loss of renal function and higher rates of cardiovascular disease in patients with chronic kidney disease. Thus, IHH connects leukocyte activation to Gli1+ cell expansion and represents a potential target for therapies to inhibit inflammation-induced fibrosis.

Senolytic treatment preserves biliary regenerative capacity lost through cellular senescence during cold storage.

Liver transplantation is the only curative option for patients with end-stage liver disease. Despite improvements in surgical techniques, nonanastomotic strictures (characterized by the progressive loss of biliary tract architecture) continue to occur after liver transplantation, negatively affecting liver function and frequently leading to graft loss and retransplantation. To study the biological effects of organ preservation before liver transplantation, we generated murine models that recapitulate liver procurement and static cold storage. In these models, we explored the response of cholangiocytes and hepatocytes to cold storage, focusing on responses that affect liver regeneration, including DNA damage, apoptosis, and cellular senescence. We show that biliary senescence was induced during organ retrieval and exacerbated during static cold storage, resulting in impaired biliary regeneration. We identified decoy receptor 2 (DCR2)-dependent responses in cholangiocytes and hepatocytes, which differentially affected the outcome of those populations during cold storage. Moreover, CRISPR-mediated DCR2 knockdown in vitro increased cholangiocyte proliferation and decreased cellular senescence but had the opposite effect in hepatocytes. Using the p21KO model to inhibit senescence onset, we showed that biliary tract architecture was better preserved during cold storage. Similar results were achieved by administering senolytic ABT737 to mice before procurement. Last, we perfused senolytics into discarded human donor livers and showed that biliary architecture and regenerative capacities were better preserved. Our results indicate that cholangiocytes are susceptible to senescence and identify the use of senolytics and the combination of senotherapies and machine-perfusion preservation to prevent this phenotype and reduce the incidence of biliary injury after transplantation.

Single-cell analysis of senescent epithelia reveals targetable mechanisms promoting fibrosis.

Progressive fibrosis and maladaptive organ repair result in significant morbidity and millions of premature deaths annually. Senescent cells accumulate with aging and after injury and are implicated in organ fibrosis, but the mechanisms by which senescence influences repair are poorly understood. Using 2 murine models of injury and repair, we show that obstructive injury generated senescent epithelia, which persisted after resolution of the original injury, promoted ongoing fibrosis, and impeded adaptive repair. Depletion of senescent cells with ABT-263 reduced fibrosis in reversed ureteric obstruction and after renal ischemia/reperfusion injury. We validated these findings in humans, showing that senescence and fibrosis persisted after relieved renal obstruction. We next characterized senescent epithelia in murine renal injury using single-cell RNA-Seq. We extended our classification to human kidney and liver disease and identified conserved profibrotic proteins, which we validated in vitro and in human disease. We demonstrated that increased levels of protein disulfide isomerase family A member 3 (PDIA3) augmented TGF-ß-mediated fibroblast activation. Inhibition of PDIA3 in vivo significantly reduced kidney fibrosis during ongoing renal injury and as such represented a new potential therapeutic pathway. Analysis of the signaling pathways of senescent epithelia connected senescence to organ fibrosis, permitting rational design of antifibrotic therapies.

The Role of Ageing and Parenchymal Senescence on Macrophage Function and Fibrosis.

In this review, we examine senescent cells and the overlap between the direct biological impact of senescence and the indirect impact senescence has via its effects on other cell types, particularly the macrophage. The canonical roles of macrophages in cell clearance and in other physiological functions are discussed with reference to their functions in diseases of the kidney and other organs. We also explore the translational potential of different approaches based around the macrophage in future interventions to target senescent cells, with the goal of preventing or reversing pathologies driven or contributed to in part by senescent cell load in vivo.

Cellular senescence inhibits renal regeneration after injury in mice, with senolytic treatment promoting repair.

The ability of the kidney to regenerate successfully after injury is lost with advancing age, chronic kidney disease, and after irradiation. The factors responsible for this reduced regenerative capacity remain incompletely understood, with increasing interest in a potential role for cellular senescence in determining outcomes after injury. Here, we demonstrated correlations between senescent cell load and functional loss in human aging and chronic kidney diseases including radiation nephropathy. We dissected the causative role of senescence in the augmented fibrosis occurring after injury in aged and irradiated murine kidneys. In vitro studies on human proximal tubular epithelial cells and in vivo mouse studies demonstrated that senescent renal epithelial cells produced multiple components of the senescence-associated secretory phenotype including transforming growth factor ß1, induced fibrosis, and inhibited tubular proliferative capacity after injury. Treatment of aged and irradiated mice with the B cell lymphoma 2/w/xL inhibitor ABT-263 reduced senescent cell numbers and restored a regenerative phenotype in the kidneys with increased tubular proliferation, improved function, and reduced fibrosis after subsequent ischemia-reperfusion injury. Senescent cells are key determinants of renal regenerative capacity in mice and represent emerging treatment targets to protect aging and vulnerable kidneys in man.

Granulocyte macrophage-colony stimulating factor: A key modulator of renal mononuclear phagocyte plasticity.

Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF) play key roles in the differentiation of macrophages and dendritic cells (DCs). We examined the effect of treatment with M-CSF-containing macrophage medium or GM-CSF-containing DC medium upon the phenotype of murine bone marrow-derived macrophages and DCs. Culture of macrophages for 5 days in DC medium reduced F4/80 expression and increased CD11c expression with cells effectively stimulating T cell proliferation in a mixed lymphocyte reaction. DC medium treatment of macrophages significantly reduced phagocytosis of both apoptotic cells and latex beads and strongly induced the expression of the chemokine receptor CCR7 known to be involved in DC trafficking to lymph nodes. Lysates of obstructed murine kidneys expressed both M-CSF and GM-CSF though M-CSF expression was dominant (M-CSF:GM-CSF ratio ∼30:1). However, combination treatment with both M-CSF and GM-CSF (ratio 30:1) indicated that small amounts of GM-CSF skewed macrophages towards a DC-like phenotype. To determine whether macrophage phenotype might be modulated in vivo we tracked CD45.1+ bone marrow-derived macrophages intravenously administered to CD45.2+ mice with unilateral ureteric obstruction. Flow cytometry of enzyme dissociated kidneys harvested 3 days later indicated CD11c and MHC Class II upregulation by adoptively transferred CD45.1+ cells with CD45.1+ cells evident in draining renal lymph nodes. Our data suggests that GM-CSF modulates mononuclear phagocyte plasticity, which likely promotes resolution of injury and healing in the injured kidney.

11ß-HSD1 suppresses cardiac fibroblast CXCL2, CXCL5 and neutrophil recruitment to the heart post MI.

We have previously demonstrated that neutrophil recruitment to the heart following myocardial infarction (MI) is enhanced in mice lacking 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) that regenerates active glucocorticoid within cells from intrinsically inert metabolites. The present study aimed to identify the mechanism of regulation. In a mouse model of MI, neutrophil mobilization to blood and recruitment to the heart were higher in 11ß-HSD1-deficient (Hsd11b1-/- ) relative to wild-type (WT) mice, despite similar initial injury and circulating glucocorticoid. In bone marrow chimeric mice, neutrophil mobilization was increased when 11ß-HSD1 was absent from host cells, but not when absent from donor bone marrow-derived cells. Consistent with a role for 11ß-HSD1 in 'host' myocardium, gene expression of a subset of neutrophil chemoattractants, including the chemokines Cxcl2 and Cxcl5, was selectively increased in the myocardium of Hsd11b1-/- mice relative to WT. SM22α-Cre directed disruption of Hsd11b1 in smooth muscle and cardiomyocytes had no effect on neutrophil recruitment. Expression of Cxcl2 and Cxcl5 was elevated in fibroblast fractions isolated from hearts of Hsd11b1-/- mice post MI and provision of either corticosterone or of the 11ß-HSD1 substrate, 11-dehydrocorticosterone, to cultured murine cardiac fibroblasts suppressed IL-1α-induced expression of Cxcl2 and Cxcl5 These data identify suppression of CXCL2 and CXCL5 chemoattractant expression by 11ß-HSD1 as a novel mechanism with potential for regulation of neutrophil recruitment to the injured myocardium, and cardiac fibroblasts as a key site for intracellular glucocorticoid regeneration during acute inflammation following myocardial injury.

Bone marrow transplantation modulates tissue macrophage phenotype and enhances cardiac recovery after subsequent acute myocardial infarction.

BACKGROUND: Bone marrow transplantation (BMT) is commonly used in experimental studies to investigate the contribution of BM-derived circulating cells to different disease processes. During studies investigating the cardiac response to acute myocardial infarction (MI) induced by permanent coronary ligation in mice that had previously undergone BMT, we found that BMT itself affects the remodelling response. METHODS AND RESULTS: Compared to matched naive mice, animals that had previously undergone BMT developed significantly less post-MI adverse remodelling, infarct thinning and contractile dysfunction as assessed by serial magnetic resonance imaging. Cardiac rupture in male mice was prevented. Histological analysis showed that the infarcts of mice that had undergone BMT had a significantly higher number of inflammatory cells, surviving cardiomyocytes and neovessels than control mice, as well as evidence of significant haemosiderin deposition. Flow cytometric and histological analyses demonstrated a higher number of alternatively activated (M2) macrophages in myocardium of the BMT group compared to control animals even before MI, and this increased further in the infarcts of the BMT mice after MI. CONCLUSIONS: The process of BMT itself substantially alters tissue macrophage phenotype and the subsequent response to acute MI. An increase in alternatively activated macrophages in this setting appears to enhance cardiac recovery after MI.

Cardiomyocyte and Vascular Smooth Muscle-Independent 11ß-Hydroxysteroid Dehydrogenase 1 Amplifies Infarct Expansion, Hypertrophy, and the Development of Heart Failure After Myocardial Infarction in Male Mice.

Global deficiency of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), an enzyme that regenerates glucocorticoids within cells, promotes angiogenesis, and reduces acute infarct expansion after myocardial infarction (MI), suggesting that 11ß-HSD1 activity has an adverse influence on wound healing in the heart after MI. The present study investigated whether 11ß-HSD1 deficiency could prevent the development of heart failure after MI and examined whether 11ß-HSD1 deficiency in cardiomyocytes and vascular smooth muscle cells confers this protection. Male mice with global deficiency in 11ß-HSD1, or with Hsd11b1 disruption in cardiac and vascular smooth muscle (via SM22α-Cre recombinase), underwent coronary artery ligation for induction of MI. Acute injury was equivalent in all groups. However, by 8 weeks after induction of MI, relative to C57Bl/6 wild type, globally 11ß-HSD1-deficient mice had reduced infarct size (34.7 ± 2.1% left ventricle [LV] vs 44.0 ± 3.3% LV, P = .02), improved function (ejection fraction, 33.5 ± 2.5% vs 24.7 ± 2.5%, P = .03) and reduced ventricular dilation (LV end-diastolic volume, 0.17 ± 0.01 vs 0.21 ± 0.01 mL, P = .01). This was accompanied by a reduction in hypertrophy, pulmonary edema, and in the expression of genes encoding atrial natriuretic peptide and ß-myosin heavy chain. None of these outcomes, nor promotion of periinfarct angiogenesis during infarct repair, were recapitulated when 11ß-HSD1 deficiency was restricted to cardiac and vascular smooth muscle. 11ß-HSD1 expressed in cells other than cardiomyocytes or vascular smooth muscle limits angiogenesis and promotes infarct expansion with adverse ventricular remodeling after MI. Early pharmacological inhibition of 11ß-HSD1 may offer a new therapeutic approach to prevent heart failure associated with ischemic heart disease.

The adult murine heart has a sparse, phagocytically active macrophage population that expands through monocyte recruitment and adopts an 'M2' phenotype in response to Th2 immunologic challenge.

Tissue resident macrophages have vital homeostatic roles in many tissues but their roles are less well defined in the heart. The present study aimed to identify the density, polarisation status and distribution of macrophages in the healthy murine heart and to investigate their ability to respond to immune challenge. Histological analysis of hearts from CSF-1 receptor (csf1-GFP; MacGreen) and CX3CR1 (Cx3cr1(GFP/+)) reporter mice revealed a sparse population of GFP positive macrophages that were evenly distributed throughout the left and right ventricular free walls and septum. F4/80+CD11b+ cardiac macrophages, sorted from myocardial homogenates, were able to phagocytose fluorescent beads in vitro and expressed markers typical of both 'M1' (IL-1ß, TNF and CCR2) and 'M2' activation (Ym1, Arg 1, RELMα and IL-10), suggesting no specific polarisation in healthy myocardium. Exposure to Th2 challenge by infection of mice with helminth parasites Schistosoma mansoni, or Heligmosomoides polygyrus, resulted in an increase in cardiac macrophage density, adoption of a stellate morphology and increased expression of Ym1, RELMα and CD206 (mannose receptor), indicative of 'M2' polarisation. This was dependent on recruitment of Ly6ChighCCR2+ monocytes and was accompanied by an increase in collagen content. In conclusion, in the healthy heart resident macrophages are relatively sparse and have a phagocytic role. Following Th2 challenge this population expands due to monocyte recruitment and adopts an 'M2' phenotype associated with increased tissue fibrosis.

Alternative activation of macrophages by filarial nematodes is MyD88-independent.

Alternative macrophage activation is largely defined by IL-4Rα stimulation but the contribution of Toll-like receptor (TLR) signaling to this phenotype is not currently known. We have investigated macrophage activation status under Th2 conditions in the absence of the core TLR adaptor molecule, MyD88. No impairment was observed in the ability of MyD88-deficient bone marrow derived macrophages to produce or express alternative activation markers, including arginase, RELM-α or Ym1, in response to IL-4 treatment in vitro. Further, we observed no difference in the ability of peritoneal exudate cells from nematode implanted wild type (WT) or MyD88-deficient mice to produce arginase or express the alternative activation markers RELM-α or Ym1. Therefore, MyD88 is not a fundamental requirement for Th2-driven macrophage alternative activation, either in vitro or in vivo.

Conditional ablation of macrophages disrupts ovarian vasculature.

Macrophages are the most abundant immune cell within the ovary. Their dynamic distribution throughout the ovarian cycle and heterogenic array of functions suggest the involvement in various ovarian processes, but their functional role has yet to be fully established. The aim was to induce conditional macrophage ablation to elucidate the putative role of macrophages in maintaining the integrity of ovarian vasculature. Using the CD11b-diphtheria toxin receptor (DTR) mouse, in which expression of human DTR is under the control of the macrophage-specific promoter sequence CD11b, ovarian macrophages were specifically ablated in adult females by injections of diphtheria toxin (DT). CD11b-DTR mice were given DT treatment or vehicle and ovaries collected at 2, 8, 16, 24 and 48  h. Histochemical stains were employed to characterise morphological changes, immunohistochemistry for F4/80 to identify macrophages and the endothelial cell marker CD31 used to quantify vascular changes. In normal ovaries, macrophages were detected in corpora lutea and in the theca layer of healthy and atretic follicles. As macrophage ablation progressed, increasing amounts of ovarian haemorrhage were observed affecting both luteal and thecal tissue associated with significant endothelial cell depletion, increased erythrocyte accumulation and increased follicular atresia by 16  h. These events were followed by necrosis and profound structural damage. Changes were limited to the ovary, as DT treatment does not disrupt the vasculature of other tissues likely reflecting the unique cyclical nature of the ovarian vasculature and heterogeneity between macrophages within different tissues. These results show that macrophages play a critical role in maintaining ovarian vascular integrity.

Macrophages and renal fibrosis.

Renal fibrosis is a key determinant of the progression of renal disease irrespective of the original cause and thus can be regarded as a final common pathway that dictates eventual outcome. The development of renal fibrosis involves many cellular and molecular mediators including leukocytes, myofibroblasts, cytokines, and growth factors, as well as metalloproteinases and their endogenous inhibitors. Study of experimental and human renal disease has shown the involvement of macrophages in renal fibrosis resulting from diverse disease processes. Recent work exploring the nature of both circulating monocytes and tissue macrophages has highlighted their multifaceted phenotype and this impacts their role in renal fibrosis in vivo. In this review we outline the key players in the fibrotic response of the injured kidney and discuss the role of monocytes and macrophages in renal scarring.

Plasmodium chabaudi limits early Nippostrongylus brasiliensis-induced pulmonary immune activation and Th2 polarization in co-infected mice.

BACKGROUND: Larvae of several common species of parasitic nematodes obligately migrate through, and often damage, host lungs. The larvae induce strong pulmonary Type 2 immune responses, including T-helper (Th)2 cells as well as alternatively activated macrophages (AAMphi) and associated chitinase and Fizz/resistin family members (ChaFFs), which are thought to promote tissue repair processes. Given the prevalence of systemic or lung-resident Type 1-inducing pathogens in geographical areas in which nematodes are endemic, we wished to investigate the impact of concurrent Type 1 responses on the development of these Type 2 responses to nematode larval migration. We therefore infected BALB/c mice with the nematode Nippostrongylus brasiliensis, in the presence or absence of Plasmodium chabaudi chabaudi malaria parasites. Co-infected animals received both infections on the same day, and disease was assessed daily before immunological measurements were taken at 3, 5, 7 or 20 days post-infection. RESULTS: We observed that the nematodes themselves caused transient loss of body mass and red blood cell density, but co-infection then slightly ameliorated the severity of malarial anaemia. We also tracked the development of immune responses in the lung and thoracic lymph node. By the time of onset of the adaptive immune response around 7 days post-infection, malaria co-infection had reduced pulmonary expression of ChaFFs. Assessment of the T cell response demonstrated that the Th2 response to the nematode was also significantly impaired by malaria co-infection. CONCLUSION: P. c. chabaudi co-infection altered both local and lymph node Type 2 immune activation due to migration of N. brasiliensis larvae. Given recent work from other laboratories showing that N. brasiliensis-induced ChaFFs correlate to the extent of long-term lung damage, our results raise the possibility that co-infection with malaria might alter pulmonary repair processes following nematode migration. Further experimentation in the co-infection model developed here will reveal the longer-term consequences of the presence of both malaria and helminths in the lung.

Alternatively activated macrophages elicited by helminth infection can be reprogrammed to enable microbial killing.

The prime function of classically activated macrophages (activated by Th1-type signals, such as IFN-gamma) is microbial destruction. Alternatively activated macrophages (activated by Th2 cytokines, such as IL-4 and IL-13) play important roles in allergy and responses to helminth infection. We utilize a murine model of filarial infection, in which adult nematodes are surgically implanted into the peritoneal cavity of mice, as an in vivo source of alternatively activated macrophages. At 3 wk postinfection, the peritoneal exudate cell population is dominated by macrophages, termed nematode-elicited macrophages (NeMphi), that display IL-4-dependent features such as the expression of arginase 1, RELM-alpha (resistin-like molecule alpha), and Ym1. Since increasing evidence suggests that macrophages show functional adaptivity, the response of NeMphi to proinflammatory Th1-activating signals was investigated to determine whether a switch between alternative and classical activation could occur in macrophages differentiated in an in vivo infection setting. Despite the long-term exposure to Th2 cytokines and antiinflammatory signals in vivo, we found that NeMphi were not terminally differentiated but could develop a more classically activated phenotype in response to LPS and IFN-gamma. This was reflected by a switch in the enzymatic pathway for arginine metabolism from arginase to inducible NO synthase and the reduced expression of RELM-alpha and Ym1. Furthermore, this enabled NeMphi to become antimicrobial, as LPS/IFN-gamma-treated NeMphi produced NO that mediated killing of Leishmania mexicana. However, the adaptation to antimicrobial function did not extend to key regulatory pathways, such as IL-12 production, which remained unaltered.

Contrasting effects of Elg1-RFC and Ctf18-RFC inactivation in the absence of fully functional RFC in fission yeast.

Proliferating cell nuclear antigen loading onto DNA by replication factor C (RFC) is a key step in eukaryotic DNA replication and repair processes. In this study, the C-terminal domain (CTD) of the large subunit of fission yeast RFC is shown to be essential for its function in vivo. Cells carrying a temperature-sensitive mutation in the CTD, rfc1-44, arrest with incompletely replicated chromosomes, are sensitive to DNA damaging agents, are synthetically lethal with other DNA replication mutants, and can be suppressed by mutations in rfc5. To assess the contribution of the RFC-like complexes Elg1-RFC and Ctf18-RFC to the viability of rfc1-44, genes encoding the large subunits of these complexes have been deleted and overexpressed. Inactivation of Ctf18-RFC by the deletion of ctf18+, dcc1+ or ctf8+ is lethal in an rfc1-44 background showing that full Ctf18-RFC function is required in the absence of fully functional RFC. In contrast, rfc1-44 elg1Delta cells are viable and overproduction of Elg1 in rfc1-44 is lethal, suggesting that Elg1-RFC plays a negative role when RFC function is inhibited. Consistent with this, the deletion of elg1+ is shown to restore viability to rfc1-44 ctf18Delta cells.