Aging modulates the effects of ischemic injury upon mesenchymal cells within the renal interstitium and microvasculature

Abstract The renal mesenchyme contains heterogeneous cells, including interstitial fibroblasts and pericytes, with key roles in wound healing. Although healing is impaired in aged kidneys, the effect of age and injury on the mesenchyme remains poorly understood. We characterized renal mesenchymal cell heterogeneity in young vs old animals and after ischemia‐reperfusion‐injury (IRI) using multiplex immunolabeling and single cell transcriptomics. Expression patterns of perivascular cell markers (α‐SMA, CD146, NG2, PDGFR‐α, and PDGFR‐β) correlated with their interstitial location. PDGFR‐α and PDGFR‐β co‐expression labeled renal myofibroblasts more efficiently than the current standard marker α‐SMA, and CD146 was a superior murine renal pericyte marker. Three renal mesenchymal subtypes; pericytes, fibroblasts, and myofibroblasts, were recapitulated with data from two independently performed single cell transcriptomic analyzes of murine kidneys, the first dataset an aging cohort and the second dataset injured kidneys following IRI. Mesenchymal cells segregated into subtypes with distinct patterns of expression with aging and following injury. Baseline uninjured old kidneys resembled post‐ischemic young kidneys, with this phenotype further exaggerated following IRI. These studies demonstrate that age modulates renal perivascular/interstitial cell marker expression and transcriptome at baseline and in response to injury and provide tools for the histological and transcriptomic analysis of renal mesenchymal cells, paving the way for more accurate classification of renal mesenchymal cell heterogeneity and identification of age‐specific pathways and targets

Kidney single-cell atlas reveals myeloid heterogeneity in progression and regression of kidney disease

Background Little is known about the roles of myeloid cell subsets in kidney injury and in the limited ability of the organ to repair itself. Characterizing these cells based only on surface markers using flow cytometry might not provide a full phenotypic picture. Defining these cells at the single-cell, transcriptomic level could reveal myeloid heterogeneity in the progression and regression of kidney disease. Methods Integrated droplet– and plate-based single-cell RNA sequencing were used in the murine, reversible, unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single-cell level during renal injury and the resolution of fibrosis. Paired blood exchange tracked the fate of monocytes recruited to the injured kidney. Results A single-cell atlas of the kidney generated using transcriptomics revealed marked changes in the proportion and gene expression of renal cell types during injury and repair. Conventional flow cytometry markers would not have identified the 12 myeloid cell subsets. Monocytes recruited to the kidney early after injury rapidly adopt a proinflammatory, profibrotic phenotype that expresses Arg1, before transitioning to become Ccr21 macrophages that accumulate in late injury. Conversely, a novel Mmp121 macrophage subset acts during repair. Conclusions Complementary technologies identified novel myeloid subtypes, based on transcriptomics in single cells, that represent therapeutic targets to inhibit progression or promote regression of kidney disease

Brief Review Renal Aging. Causes and consequences

Individuals age >65 years old are the fastest expanding population demographic throughout the developed world. Consequently, more aged patients than before are receiving diagnoses of impaired renal function and nephrosclerosis—age–associated histologic changes in the kidneys. Recent studies have shown that the aged kidney undergoes a range of structural changes and has altered transcriptomic, hemodynamic, and physiologic behavior at rest and in response to renal insults. These changes impair the ability of the kidney to withstand and recover from injury, contributing to the high susceptibility of the aged population to AKI and their increased propensity to develop subsequent progressive CKD. In this review, we examine these features of the aged kidney and explore the various validated and putative pathways contributing to the changes observed with aging in both experimental animal models and humans. We also discuss the potential for additional study to increase understanding of the aged kidney and lead to novel therapeutic strategies

Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis

Although macrophages are widely recognized to have a profibrotic role in inflammation, we have used a highly tractable CCl(4)-induced model of reversible hepatic fibrosis to identify and characterize the macrophage phenotype responsible for tissue remodeling: the hitherto elusive restorative macrophage. This CD11B(hi) F4/80(int) Ly-6C(lo) macrophage subset was most abundant in livers during maximal fibrosis resolution and represented the principle matrix metalloproteinase (MMP) -expressing subset. Depletion of this population in CD11B promoter–diphtheria toxin receptor (CD11B-DTR) transgenic mice caused a failure of scar remodeling. Adoptive transfer and in situ labeling experiments showed that these restorative macrophages derive from recruited Ly-6C(hi) monocytes, a common origin with profibrotic Ly-6C(hi) macrophages, indicative of a phenotypic switch in vivo conferring proresolution properties. Microarray profiling of the Ly-6C(lo) subset, compared with Ly-6C(hi) macrophages, showed a phenotype outside the M1/M2 classification, with increased expression of MMPs, growth factors, and phagocytosis-related genes, including Mmp9, Mmp12, insulin-like growth factor 1 (Igf1), and Glycoprotein (transmembrane) nmb (Gpnmb). Confocal microscopy confirmed the postphagocytic nature of restorative macrophages. Furthermore, the restorative macrophage phenotype was recapitulated in vitro by the phagocytosis of cellular debris with associated activation of the ERK signaling cascade. Critically, induced phagocytic behavior in vivo, through administration of liposomes, increased restorative macrophage number and accelerated fibrosis resolution, offering a therapeutic strategy to this orphan pathological process

A mutant O-GlcNAcase enriches Drosophila developmental regulators

YesProtein O-GlcNAcylation is a reversible post-translational modification of serines/threonines on nucleocytoplasmic proteins. It is cycled by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). Genetic approaches in model organisms have revealed that protein O-GlcNAcylation is essential for early embryogenesis. Drosophila melanogaster OGT/supersex combs (sxc) is a polycomb gene, null mutants of which display homeotic transformations and die at the pharate adult stage. However, the identities of the O-GlcNAcylated proteins involved, and the underlying mechanisms linking these phenotypes to embryonic development, are poorly understood. Identification of O-GlcNAcylated proteins from biological samples is hampered by the low stoichiometry of this modification and limited enrichment tools. Using a catalytically inactive bacterial O-GlcNAcase mutant as a substrate trap, we have enriched the O-GlcNAc proteome of the developing Drosophila embryo, identifying, amongst others, known regulators of Hox genes as candidate conveyors of OGT function during embryonic development.Wellcome Trust Investigator Award (110061); MRC grant (MC_UU_12016/5); and Royal Society Research Grant

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-β–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

Apoptotic cell administration is detrimental in murine renal ischaemia reperfusion injury

BACKGROUND: Acute kidney injury induced by renal ischaemia reperfusion injury (IRI) is characterised by renal failure, acute tubular necrosis (ATN), inflammation and microvascular congestion. The administration of apoptotic cells (ACs) has been shown to reduce inflammation in various organs including the liver and kidney. This study explored whether AC administration prior to the induction of renal IRI was protective. FINDINGS: Renal IRI was induced in Balb/c mice by clamping the renal blood vessels for either 20, 24 or 25 minutes to induce mild, moderate or severe kidney dysfunction respectively. Renal function and injury was determined 24 hours following IRI by measurement of plasma creatinine and ATN scoring respectively. ACs were generated from Balb/c thymocytes and classified as either predominantly early or late apoptotic by Annexin-V and propidium iodide staining. Early AC administration prior to severe IRI had no influence on plasma creatinine or ATN severity. In contrast, administration of early or late ACs significantly worsened renal function in mice with mild or moderate renal IRI, respectively, compared to PBS treated controls, though ATN scores were comparable. Despite ACs exerting pro-coagulant effects, the worsening of renal function was not secondary to increased microvascular congestion, inferred by fibrin and platelet (CD41) deposition, or inflammation, assessed by neutrophil infiltration. CONCLUSIONS: Despite the AC-derived protection demonstrated in other organs, ACs do not protect mice from renal IRI. ACs may in fact further impair renal function depending on injury severity. These data suggest that AC-derived protection is not translationally relevant for patients with acute kidney injury induced by ischaemic injury. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12950-014-0031-6) contains supplementary material, which is available to authorized users

The role of epigenetics in renal ageing

An ability to separate natural ageing processes from processes specific to morbidities is required to understand the heterogeneity of age-related organ dysfunction. Mechanistic insight into how epigenetic factors regulate ageing throughout the life course, linked to a decline in renal function with ageing, is already proving to be of value in the analyses of clinical and epidemiological cohorts. Noncoding RNAs provide epigenetic regulatory circuits within the kidney, which reciprocally interact with DNA methylation processes, histone modification and chromatin. These interactions have been demonstrated to reflect the biological age and function of renal allografts. Epigenetic factors control gene expression and activity in response to environmental perturbations. They also have roles in highly conserved signalling pathways that modulate ageing, including the mTOR and insulin/insulin-like growth factor signalling pathways, and regulation of sirtuin activity. Nutrition, the gut microbiota, inflammation and environmental factors, including psychosocial and lifestyle stresses, provide potential mechanistic links between the epigenetic landscape of ageing and renal dysfunction. Approaches to modify the renal epigenome via nutritional intervention, targeting the methylome or targeting chromatin seem eminently feasible, although caution is merited owing to the potential for intergenerational and transgenerational effects