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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.

Kidney disease represents a global health burden of increasing prevalence and is an independent risk factor for cardiovascular disease. Myeloid cells are a major cellular compartment of the immune system; they are found in the healthy kidney and in increased numbers in the damaged and/or diseased kidney, where they act as key players in the progression of injury, inflammation and fibrosis. They possess enormous plasticity and heterogeneity, adopting different phenotypic and functional characteristics in response to stimuli in the local milieu. Though this inherent complexity remains to be fully understood in the kidney, advances in single-cell genomics promises to change this. Specifically, single-cell RNA sequencing (scRNA-seq) has had a transformative effect on kidney research, enabling the profiling and analysis of the transcriptomes of single cells at unprecedented resolution and throughput, and subsequent generation of cell atlases. Moving forward, combining scRNA- and single-nuclear RNA-seq with greater resolution spatial transcriptomics will allow spatial mapping of kidney disease of varying aetiology to further reveal the patterning of immune cells and non-immune renal cells.This review summarises the roles of myeloid cells in kidney health and disease, the experimental workflow in currently available scRNA-seq technologies and published findings using scRNA-seq in the context of myeloid cells and the kidney.

Dyslipidemia is a main driver of cardiovascular diseases. The ability of macrophages to scavenge excess lipids implicate them as mediators in this process and understanding the mechanisms underlying macrophage lipid metabolism is key to the development of new treatments. Here, we investigated how adipose tissue macrophages regulate post-prandial cholesterol transport. Single-cell RNA sequencing and protected bone marrow chimeras demonstrated that ingestion of lipids led to specific transcriptional activation of a population of resident macrophages expressing Lyve1, Tim4, and ABCA1. Blocking the phosphatidylserine receptor Tim4 inhibited lysosomal activation and the release of post-prandial high density lipoprotein cholesterol following a high fat meal. Both effects were recapitulated by chloroquine, an inhibitor of lysosomal function. Moreover, clodronate-mediated cell-depletion implicated Tim4+ resident adipose tissue macrophages in this process. Thus, these data indicate that Tim4 is a key regulator of post-prandial cholesterol transport and adipose tissue macrophage function and may represent a novel pathway to treat dyslipidemia., Diverse macrophage subsets are found in adipose tissue where they regulate its physiology. Here, the authors used single-cell RNA sequencing to analyse the effect of post-prandial lipids on adipose tissue macrophages and identify Tim4 as a regulator of ABCA1+ macrophage function and post-prandial cholesterol transport.

Rationale: In the microenvironment of atherosclerotic lesions, vascular smooth muscle cells (vSMCs) switch to a dedifferentiated state but the underlying molecular mechanisms driving this switch are not fully understood. Long noncoding RNAs (lncRNAs) are dysregulated during vascular pathology, but relatively little is known about their involvement in controlling vSMCs function. Cardiac mesoderm enhancer-associated noncoding RNA (CARMN) is a lncRNA located immediately upstream of the microRNAs-143 and -145 (miR-143 and miR-145 ), both involved in vSMCs function. Objective: We investigated the role of the lncRNA CARMN, independent from miR-143 and miR-145, as a potential regulator of vSMC phenotypes in vitro and the consequences of its loss during the development of atherosclerosis in vivo. We hypothesized that loss of CARMN is a primary event controlling the functional switch towards proatherogenic vSMC phenotype and accelerates the development of the plaques in vivo. Method and Results: Expression of CARMN lncRNA was silenced using locked nucleic acids antisense oligonucleotides (GapmeRs) in human coronary arterial smooth muscle cells, revealing that GapmeR-mediated loss of CARMN negatively affects miR-143 and miR-145 microRNA expression. RNA sequencing of CARMN-depleted human coronary arterial smooth muscle cells revealed large transcriptomic changes, associated with vSMC proliferation, migration, inflammation, lipid metabolism, and dedifferentiation. The use of miR-143 and miR-145 mimics revealed that CARMN regulates human coronary arterial smooth muscle cell proliferation in a microRNA-independent manner. In humans and mice, CARMN and associated microRNAs were downregulated in advanced versus early atherosclerotic lesions. Using a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) knockout approach, we explored the implications of CARMN depletion during atherosclerosis in vivo. Consistent with in vitro results, the knockout of CARMN impaired the expression of miR-143 and miR-145 under homeostatic conditions. Importantly, when atherosclerosis was induced in these mice, CARMN knockout increased the volume, size, proinflammatory Lgals3 (galectin 3)-expressing cells content, and altered plaque composition, yielding an advanced phenotype. Conclusions: We identified the early loss of CARMN lncRNA as critical event which primes vSMCs towards a proatherogenic phenotype in vitro and accelerates the development of atherosclerosis in vivo.

Progressive fibrosis and maladaptive organ repair result in significant morbidity and millions of premature deaths annually. Senescent cells accumulate with ageing and after injury and are implicated in organ fibrosis, but the mechanisms by which senescence influences repair are poorly understood. Using two murine models of injury and repair we show that obstructive injury generates senescent epithelia which persist after resolution of the original injury, promote ongoing fibrosis and impede adaptive repair. Depletion of senescent cells with ABT263 reduces fibrosis in reversed ureteric obstruction and after renal ischaemia-reperfusion injury. We validate these findings in humans, showing that senescence and fibrosis persist after relieved renal obstruction. We next characterise senescent epithelia in murine renal injury using single cell RNA-Seq. We extend our classification to human kidney and liver disease and identify conserved pro-fibrotic proteins which we validate in vitro and in human disease. We demonstrate that one such molecule, Protein Disulfide Isomerase Family A Member 3 (PDIA3), is essential for TGF-beta mediated fibroblast activation. Inhibition of PDIA3 in vivo significantly reduces kidney fibrosis during ongoing renal injury and as such represents a new potential therapeutic pathway. Analysis of the signalling pathways of senescent epithelia connects senescence to organ fibrosis, permitting rational design of anti-fibrotic therapies.

Ultrasound has previously been demonstrated to non-invasively cause tissue disruption. Small animal studies have demonstrated that this effect can be enhanced by contrast microbubbles and has the potential to be clinically beneficial in techniques such as targeted drug delivery or enhancing liquid biopsies when a physical biopsy may be inappropriate. Cavitating microbubbles in close proximity to cells increases membrane permeability, allowing small intracellular molecules to leak into the extracellular space. This study sought to establish whether cavitating microbubbles could liberate cell-specific miRNAs, augmenting biomarker detection for non-invasive liquid biopsies. Insonating human polarized renal proximal tubular epithelial cells (RPTECs), in the presence of SonoVue microbubbles, revealed that cellular health could be maintained while achieving the release of miRNAs, miR-21, miR-30e, miR-192 and miR-194 (respectively, 10.9-fold, 7.17-fold, 5.95-fold and 5.36-fold). To examine the mechanism of release, RPTECs expressing enhanced green fluorescent protein were generated and the protein successfully liberated. Cell polarization, cellular phenotype and cell viability after sonoporation were measured by a number of techniques. Ultrastructural studies using electron microscopy showed gap-junction disruption and pore formation on cellular surfaces. These studies revealed that cell-specific miRNAs can be non-specifically liberated from RPTECs by sonoporation without a significant decrease in cell viability.

Supplemental Digital Content is available in the text., GPR81 (G-protein-coupled receptor 81) is highly expressed in adipocytes, and activation by the endogenous ligand lactate inhibits lipolysis. GPR81 is also expressed in the heart, liver, and kidney, but roles in nonadipose tissues are poorly defined. GPR81 agonists, developed to improve blood lipid profile, might also provide insights into GPR81 physiology. Here, we assessed the blood pressure and renal hemodynamic responses to the GPR81 agonist, AZ′5538. In male wild-type mice, intravenous AZ′5538 infusion caused a rapid and sustained increase in systolic and diastolic blood pressure. Renal artery blood flow, intrarenal tissue perfusion, and glomerular filtration rate were all significantly reduced. AZ′5538 had no effect on blood pressure or renal hemodynamics in Gpr81−/− mice. Gpr81 mRNA was expressed in renal artery vascular smooth muscle, in the afferent arteriole, in glomerular and medullary perivascular cells, and in pericyte-like cells isolated from kidney. Intravenous AZ′5538 increased plasma ET-1 (endothelin 1), and pretreatment with BQ123 (endothelin-A receptor antagonist) prevented the pressor effects of GPR81 activation, whereas BQ788 (endothelin-B receptor antagonist) did not. Renal ischemia-reperfusion injury, which increases renal extracellular lactate, increased the renal expression of genes encoding ET-1, KIM-1 (Kidney Injury Molecule 1), collagen type 1-α1, TNF-α (tumor necrosis factor-α), and F4/80 in wild-type mice but not in Gpr81−/− mice. In summary, activation of GPR81 in vascular smooth muscle and perivascular cells regulates renal hemodynamics, mediated by release of the potent vasoconstrictor ET-1. This suggests that lactate may be a paracrine regulator of renal blood flow, particularly relevant when extracellular lactate is high as occurs during ischemic renal disease.

Supplemental Digital Content is available in the text., Rationale: Despite increasing understanding of the prognostic importance of vascular stiffening linked to perivascular fibrosis in hypertension, the molecular and cellular regulation of this process is poorly understood. Objectives: To study the functional role of microRNA-214 (miR-214) in the induction of perivascular fibrosis and endothelial dysfunction driving vascular stiffening. Methods and Results: Out of 381 miRs screened in the perivascular tissues in response to Ang II (angiotensin II)-mediated hypertension, miR-214 showed the highest induction (8-fold, P=0.0001). MiR-214 induction was pronounced in perivascular and circulating T cells, but not in perivascular adipose tissue adipocytes. Global deletion of miR-214−/− prevented Ang II-induced periaortic fibrosis, Col1a1, Col3a1, Col5a1, and Tgfb1 expression, hydroxyproline accumulation, and vascular stiffening, without difference in blood pressure. Mechanistic studies revealed that miR-214−/− mice were protected against endothelial dysfunction, oxidative stress, and increased Nox2, all of which were induced by Ang II in WT mice. Ang II-induced recruitment of T cells into perivascular adipose tissue was abolished in miR-214−/− mice. Adoptive transfer of miR-214−/− T cells into RAG1−/− mice resulted in reduced perivascular fibrosis compared with the effect of WT T cells. Ang II induced hypertension caused significant change in the expression of 1380 T cell genes in WT, but only 51 in miR-214−/−. T cell activation, proliferation and chemotaxis pathways were differentially affected. MiR-214−/− prevented Ang II-induction of profibrotic T cell cytokines (IL-17, TNF-α, IL-9, and IFN-γ) and chemokine receptors (CCR1, CCR2, CCR4, CCR5, CCR6, and CXCR3). This manifested in reduced in vitro and in vivo T cell chemotaxis resulting in attenuation of profibrotic perivascular inflammation. Translationally, we show that miR-214 is increased in plasma of patients with hypertension and is directly correlated to pulse wave velocity as a measure of vascular stiffness. Conclusions: T-cell–derived miR-214 controls pathological perivascular fibrosis in hypertension mediated by T cell recruitment and local profibrotic cytokine release.

Chronic kidney disease (CKD) and ageing inhibit tissue regeneration and increase risks of organ fibrosis and cardiovascular disease. Increased numbers of leukocytes are present in the circulation and within the kidney of ageing individuals and patients with CKD where they correlate with progressive fibrosis. The involvement of activated leukocytes in progressive renal and systemic fibrosis remains incompletely understood. Here we show that renal leukocyte derived tumour necrosis factor alpha (TNFα) promotes renal and cardiac fibrosis via downstream induction of Indian Hedgehog (IHH). We identify the Ubiquitin D expressing ‘inflammatory’ proximal tubular epithelia (iPT) population responsible for TNFα/NFκB induced IHH production. iPT cells are upregulated in the kidney in experimental murine and human renal disease and ageing. iPT derived IHH activates canonical Hedgehog signalling in Gli1+ stromal cells, inducing their activation, proliferation and fibrosis in the surrounding kidney and in remote organs including the heart. This can be inhibited by selective genetic Ihh deletion from Pax8 expressing renal epithelia, or pharmacological blockade of TNFα, NFκB or Hedgehog signalling. This data connects inflammation to progressive renal and cardiac fibrosis and identifies new targets for anti-fibrotic therapies.

Background and Aims Fifty years of heart failure research has shown that pathological cardiac remodelling forms a vicious cycle with myocardial dysfunction leading to progressive heart failure (HF) [Circulation, 102 IV14-23, 2000]. Fetal gene induction is associated with this process and beta blocker therapy has been shown to prevent it. Although chronic kidney disease (CKD) and HF share similar mediators of cardiac remodelling, the benefits of beta blocker therapy in CKD has not been studied. We, therefore, tested the hypothesis that beta blocker therapy prevents fetal gene induction and pathological cardiac remodelling in experimental uraemia. Method Wistar rats (n=32) had subtotal nephrectomy (STNx) [Frontiers in physiology, 10 1365, 2019] or sham surgery and were followed up for 10 weeks. The animals were randomly allocated to metoprolol (10mg/kg/day) or vehicle. In vivo and in vitro cardiac assessments were performed, and changes in myocardial fetal gene expression were also studied. Results Heart rate was significantly lower in metoprolol groups compared to untreated groups demonstrating effective beta blockade (Fig 1A). Echocardiographic LV mass was significantly higher in untreated STNx group compared to the metoprolol group (896.4 vs 632.2g, P=0.0004). Similar changes were seen with heart weight to tibia ratio (Fig 1B). There was no significant difference in blood pressure (BP) between treated and untreated STNx animals (123 vs 119 mmHg, P=0.359) (Fig 1A). STNx increased mRNA expression of fetal genes and there was a trend towards attenuation of this increase with beta blocker therapy (Fig 1C). Conclusion Beta blocker therapy ameliorates uraemic pathological cardiac remodelling irrespective of changes to BP. This benefit appears be associated with a reduction of induced fetal gene expression. Further translational research on the benefits of beta blockade in the treatment of uraemic cardiomyopathy is required.

Introduction MicroRNAs are promising biomarkers of renal disease, however the cellular origin of their expression is usually unclear limiting their interpretation when measured in renal biopsies and urine. We hypothesised that by first defining renal cell-enriched microRNAs, we could select biomarkers based on the expected histopathological profile. Method Small RNA-sequencing of cortical, proximal tubular (LTL), macrophage (F480), endothelial (CD31) and fibroblast (PDGFRb) populations from the reversible unilateral ureteric obstruction (rUUO) murine model was performed. Hierarchical clustering was used to identify clusters. Findings were translated into an ischaemia reperfusion injury (IRI) model and then into urine samples from renal transplant recipients (n=16) with delayed graft function (DGF) vs. those with primary function. Result Kidney injury resulted in significant macrophage infiltration and tubular injury which improved upon reversal. We characterised novel microRNA clusters enriched for each cell type. With injury there was a significant increase in macrophage (p Conclusion This is the first study to characterise cell-enriched microRNAs during renal injury and repair. By defining the source of microRNA expression we were able to rationally select miR-16 and miR-18a as promising urinary biomarkers of renal injury. Take-home message We have found that microRNAs have differences in expression between cell types and renal injury states which is important when considering microRNA expression in samples composed of varying cellular composition. By defining the cellular origins of microRNA expression we were able to rationally select microRNA biomarkers of human renal injury.

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 Ccr2+ macrophages that accumulate in late injury. Conversely, a novel Mmp12+ 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.

Small noncoding RNA, microRNAs (miRNAs), are emerging as important modulators in the pathogenesis of kidney disease, with potential as biomarkers of kidney disease onset, progression or possibly outcome from treatment. Bulk tissue small RNA-Sequencing (sRNA-seq) and microarrays are widely used to identify dysregulated miRNA expression but are limited by the lack of precision regarding the cellular origin of the miRNA. In this study, we performed cell-specific sRNA-seq on tubular cells, endothelial cells, fibroblasts and macrophages isolated from the injured and repairing kidneys of the murine reversible unilateral ureteric obstruction model. We devised an unbiased bioinformatics pipeline to define the miRNA enrichment within these cell populations, constructing a miRNA catalogue of injury and repair. Our analysis reveals that a significant proportion of cell-specific miRNAs in healthy animals were no longer specific following injury. We then apply this knowledge of the relative cell specificity of miRNAs to deconvolute bulk miRNA expression changes in kidney disease from murine models and human renal disease kidney samples comprised of mixed cell types. Finally, we use our data-driven approach to rationally select and demonstrate macrophage enriched miR-16-5p and miR-18a as promising urinary biomarkers of acute kidney injury in renal transplant recipients.

The kidney has a limited capacity to repair following injury, however, the endogenous reparative pathways are not well understood. Here we employ integrated droplet- and plate-based scRNA-seq in the murine reversible unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single cell level during renal injury and resolution of fibrosis. We generate a comprehensive catalogue of the changes induced during injury and repair, revealing significant myeloid cell heterogeneity, which would not have been identifiable by conventional flow cytometry. We identify new markers for the myeloid populations within the kidney as well as identification of novel subsets including an Arg1+ monocyte population specific to early injury and a Mmp12+ macrophage subset exclusive to repair. Finally, using paired blood exchange to track circulating immune cells, we confirm that monocytes are recruited to the kidney early after injury and are the source of Ccr2+ macrophages that accumulate in late injury. Our data demonstrate the utility of complementary technologies to identify novel myeloid subtypes that may represent therapeutic targets to inhibit progression or promote regression of kidney disease.

Extracellular microRNAs have been demonstrated to have the ability to enter kidney tubular cells and modify gene expression. We have used a Dicer-hepatocyte-specific microRNA conditional knock-out (Dicer-CKO) mouse to investigate functional microRNA transfer from liver to kidney under physiological conditions and in the context of drug toxicity. Dicer-CKO mice demonstrated a time-dependent decrease in the hepatocyte-derived microRNA, miR-122, in the kidney in the absence of other microRNA changes. During hepatotoxicity, miR-122 increased in kidney tubular cells; this was abolished in Dicer-CKO mice. Depletion of hepatocyte microRNAs increased expression and activity of the miR-122 target - cytochrome (CYP) P450 2E1 - in the kidney. Serum extracellular vesicles (ECVs) from mice with hepatotoxicity increased proximal tubular cell miR-122 and prevented cisplatin proximal tubular cell toxicity. miR-122 also increased in urinary ECVs during hepatotoxicity in humans. Transfer of microRNA was not restricted to liver injury – we detected miR-499 release with murine cardiac injury, and this correlated with an increase in the kidney. In summary, a physiological transfer of microRNA to the kidney exists, which is increased by liver injury. Regulation of renal drug response due to signalling by microRNA of hepatic origin represents a new paradigm for understanding and preventing nephrotoxicity.

Chronic kidney disease (CKD) is prevalent worldwide and is associated with significant co-morbidities including cardiovascular disease. Traditionally, the subtotal nephrectomy (remnant kidney) experimental model has been performed in rats to model progressive renal disease. The model experimentally mimics CKD by reducing nephron number, resulting in renal insufficiency. Presently, there is a lack of translation of pre-clinical findings into successful clinical results. The pre-clinical nephrology field would benefit from reproducible progressive renal disease models in mice in order to avail of more widely available transgenics and experimental tools to dissect mechanisms of disease. Here we evaluate if a simplified single step subtotal nephrectomy (STNx) model performed in the 129S2/SV mouse can recapitulate the renal and cardiac changes observed in patients with CKD in a reproducible and robust way. The single step STNx surgery was well tolerated and resulted in clinically relevant outcomes including hypertension, increased urinary albumin:creatinine ratio, and significantly increased serum creatinine, phosphate and BUN. STNx mice developed significant left ventricular hypertrophy without reduced ejection fraction or cardiac fibrosis. Analysis of intra-renal inflammation revealed persistent recruitment of Ly6chi monocytes transitioning to pro-fibrotic inflammatory macrophages in STNx kidneys. Unlike 129S2/SV mice, C57BL/6 mice exhibited renal fibrosis without proteinuria, renal dysfunction renal disease or cardiac pathology. Therefore, the 129S2/SV genetic background is susceptible to induction of progressive proteinuric renal disease and cardiac hypertrophy using our refined, single-step flank STNx method. This reproducible model could be used to study the systemic pathophysiological changes induced by CKD in the kidney and the heart, intra-renal inflammation and for testing new therapies for CKD.

Background Excessive TGF-β signalling has been shown to underlie pulmonary hypertension (PAH). Human pulmonary artery smooth muscle cells (HPASMCs) can release extracellular vesicles (EVs) but their contents and significance have not yet been studied. Here, we aimed to analyse the contents and biological relevance of HPASMC-EVs and their transport to human pulmonary arterial endothelial cells (HPAECs), as well as the potential alteration of these under pathological conditions. Methods We used low-input RNA-Seq to analyse the RNA cargoes sorted into released HPASMC-EVs under basal conditions. We additionally analysed the effects of excessive TGF-β signalling, using TGF-β1 and BMP4, in the transcriptome of HPASMCs and their EVs. We then, for the first time, optimised Cre-loxP technology for its use with primary cells in vitro, directly visualising HPASMC-to-HPAEC communication and protein markers on cells taking up EVs. Furthermore we could analyse alteration of this transport with excessive TGF-β signalling, as well as by other cytokines involved in PAH: IL-1β, TNF-α and VEGFA. Results We were able to detect transcripts from 2417 genes in HPASMC-EVs. Surprisingly, among the 759 enriched in HPASMC-EVs compared to their donor cells, we found Zeb1 and 2 TGF-β superfamily ligands, GDF11 and TGF-β3. Moreover, we identified 90 genes differentially expressed in EVs from cells treated with TGF-β1 compared to EVs in basal conditions, including a subset involved in actin and ECM remodelling, among which were bHLHE40 and palladin. Finally, using Cre-loxP technology we showed cell-to-cell transfer and translation of HPASMC-EV Cre mRNA from HPASMC to HPAECs, effectively evidencing communication via EVs. Furthermore, we found increased number of smooth-muscle actin positive cells on HPAECs that took up HPASMC-EVs. The uptake and translation of mRNA was also higher in activated HPAECs, when stimulated with TGF-β1 or IL-1β. Conclusions HPASMC-EVs are enriched in RNA transcripts that encode genes that could contribute to vascular remodelling and EndoMT during development and PAH, and TGF-β1 up-regulates some that could enhance this effects. These EVs are functionally transported, increasingly taken up by activated HPAECs and contribute to EndoMT, suggesting a potential effect of HPASMC-EVs in TGF-β signalling and other related processes during PAH development.

The omentum is a visceral adipose tissue rich in fat-associated lymphoid clusters (FALCs) that collects peritoneal contaminants and provides a first layer of immunological defense within the abdomen. Here, we investigated the mechanisms that mediate the capture of peritoneal contaminants during peritonitis. Single-cell RNA sequencing and spatial analysis of omental stromal cells revealed that the surface of FALCs were covered by CXCL1+ mesothelial cells, which we termed FALC cover cells. Blockade of CXCL1 inhibited the recruitment and aggregation of neutrophils at FALCs during zymosan-induced peritonitis. Inhibition of protein arginine deiminase 4, an enzyme important for the release of neutrophil extracellular traps, abolished neutrophil aggregation and the capture of peritoneal contaminants by omental FALCs. Analysis of omental samples from patients with acute appendicitis confirmed neutrophil recruitment and bacterial capture at FALCs. Thus, specialized omental mesothelial cells coordinate the recruitment and aggregation of neutrophils to capture peritoneal contaminants.

Accurate biomarkers that both predict the progression to, and detect the early stages of chronic kidney disease (CKD) are lacking, resulting in difficulty in identifying individuals who could potentially benefit from targeted intervention. In a recent issue [Clinical Science (2018) 132, 2121–2133], Cui et al. examine the ability of urinary angiotensinogen (uAGT) to predict the progression of acute kidney injury (AKI) to CKD. They principally employ a murine ischaemia reperfusion injury model to study this and provide data from a small prospective study of patients with biopsy proven acute tubular necrosis. The authors suggest that uAGT is a dynamic marker of renal injury that could be used to predict the likelihood of structural recovery following AKI. Here we comment on their findings, exploring the clinical utility of uAGT as a biomarker to predict AKI to CKD transition and perhaps more controversially, to discuss whether the early renin–angiotensin system blockade following AKI represents a therapeutic target.

Objective: Hypertension (HT) is associated with perivascular inflammation and increased vascular fibrosis. MicroRNAs (miR) are a novel gene expression regulation mechanism and play a pivotal role in a range of pathological processes. The role and mechanism of miR214 in vascular fibrosis is unknown. Methods: 3-month-old C57BL/6, miR214KO and wild-type littermates were treated with angiotensin II (AngII, 490ng/kg/min; n=6-10) or control buffer for 14 days. PVATs from C57BL/6 animals were analysed using TaqMan_Rodent_microRNA_Arrays. Histological studies, wire myography, lucigenin-enhanced luminometry and cytometrical analysis was conducted, followed by statistical analysis with ANOVA or t-test. Data are expressed as a mean±SEM. Results: Out of 381 miRs, 16 were significantly overexpressed in C57BL/6 AngII animals, with only miR214 showing 8-fold induction (p Conclusions: MiR-214 plays a major role in modulation of aortic fibrosis, vascular function, oxidative stress and perivascular inflammation.

Background: Small non-coding microRNAs (miR) have important regulatory roles and are used as biomarkers of\ud disease. We investigated the relationship between lipoproteins and circulating miR-30c, evaluated how they are\ud transported in circulation and determined whether statins altered the circulating concentration of miR-30c.\ud Methods: To determine the relationship between lipoproteins and circulating miR-30c, serum samples from 79\ud subjects recruited from a lipid clinic were evaluated. Ultracentrifugation and nanoparticle tracking analysis\ud was used to evaluate the transportation of miR-30c in the circulation by lipoproteins and extracellular vesicles\ud in three healthy volunteers. Using archived samples from previous studies, the effects of 40 mg rosuvastatin\ud (n = 22) and 40 mg pravastatin (n = 24) on miR-30c expression was also examined. RNA extraction, reverse\ud transcription-quantitative real-time polymerase chain reaction was carried out using standard procedures.\ud Results: When stratified according to total cholesterol concentration, there was increased miR-30c expression in\ud the highest compared to the lowest tertile (p = 0.035). There was significant positive correlation between miR-\ud 30c and total- (r = 0.367; p = 0.002) and LDL-cholesterol (r = 0.391; p = 0.001). We found that miR-30c was\ud transported in both exosomes and on HDL3. There was a 3.8-fold increased expression of circulating miR-30c\ud after pravastatin treatment for 1 year (p = 0.005) but no significant change with atorvastatin after 8 weeks\ud (p = 0.145).\ud Conclusions: This study shows for the first-time in humans that circulating miR-30c is significantly, positively correlated\ud with total- and LDL-cholesterol implicating regulatory functions in lipid homeostasis. We show miR-30c\ud is transported in both exosomes and on HDL3 and pravastatin therapy significantly increased circulating miR-30c\ud expression adding to the pleiotropic dimensions of statins.

The interaction of human adenovirus (HAdV) serotype 5 (HAdV-5) with the blood coagulation factor X (FX) results in a high liver transduction after AdV intravascular administration, causing toxicity and limiting AdV delivery to the target tissue. However, FX also protects adenoviral vectors from neutralization by the complement system and natural antibodies, potentially benefiting adenoviral gene delivery, as neutralization results in the reduction of HAdV-5 binding to host cells.This review gives an overview on the use of adenoviruses as gene transfer vehicles and their impact in gene therapy. The structure of coagulation factors and their interactions with HAdV are described, highlighting their influence on the AdV biodistribution profile. The implications of this interaction in immunity and its potential influence on the use of adenoviral vectors in gene therapy applications are discussed.The protective role of FX brings under discussion how beneficial abolishment of FX binding would be for HAdV-5 liver detargeting. The dispensability of FX for liver transduction in immunocompromised mice suggests the involvement of other blood factors or receptors in enhancing HAdV-5 liver transduction. Better understanding of the interactions that take place in the bloodstream is essential to generate safe and efficient adenoviral vectors.

Human adenoviral serotype 5 (HAdV-5) vectors have predominantly hepatic tropism when delivered intravascularly, resulting in immune activation and toxicity. Coagulation factor X (FX) binding to HAdV-5 mediates liver transduction and provides protection from virion neutralization in mice. FX is dispensable for liver transduction in mice lacking IgM antibodies or complement, suggesting that alternative transduction pathways exist. To identify novel factor(s) mediating HAdV-5 FX-independent entry, we investigated HAdV-5 transduction in vitro in the presence of serum from immunocompetent C57BL/6 or immunocompromised mice lacking IgM antibodies (Rag 2−/− and NOD-scid-gamma [NSG]). Sera from all three mouse strains enhanced HAdV-5 transduction of A549 cells. While inhibition of HAdV-5–FX interaction with FX-binding protein (X-bp) inhibited transduction in the presence of C57BL/6 serum, it had negligible effect on the enhanced transduction observed in the presence of Rag 2−/− or NSG serum. Rag 2−/− serum also enhanced transduction of the FX binding-deficient HAdV-5HVR5*HVR7*E451Q (AdT*). Interestingly, Rag 2−/− serum enhanced HAdV-5 transduction in a FX-independent manner in CHO-CAR and SKOV3-CAR cells (CHO or SKOV3 cells transfected to stably express human coxsackievirus and adenovirus receptor [CAR]). Additionally, blockade of CAR with soluble HAdV-5 fiber knob inhibited mouse serum-enhanced transduction in A549 cells, suggesting a potential role for CAR. Transduction of HAdV-5 KO1 and HAdV-5/F35 (CAR binding deficient) in the presence of Rag 2−/− serum was equivalent to that of HAdV-5, indicating that direct interaction between HAdV-5 and CAR is not required. These data suggest that FX may protect HAdV-5 from neutralization but has minimal contribution to HAdV-5 transduction in the presence of immunocompromised mouse serum. Alternatively, transduction occurs via an unidentified mouse serum protein capable of bridging HAdV-5 to CAR. IMPORTANCE The intravascular administration of HAdV-5 vectors can result in acute liver toxicity, transaminitis, thrombocytopenia, and injury to the vascular endothelium, illustrating challenges yet to overcome for HAdV-5-mediated systemic gene therapy. The finding that CAR and potentially an unidentified factor present in mouse serum might be important mediators of HAdV-5 transduction highlights that a better understanding of the complex biology defining the interplay between adenovirus immune recognition and cellular uptake mechanisms is still required. These findings are important to inform future optimization and development of HAdV-5-based adenoviral vectors for gene therapy.

Fibrosis pathophysiology is critically regulated by Smad 2– and Smad 3–mediated transforming growth factor-β (TGF-β) signaling. Disintegrin metalloproteases (Adam) can manipulate the signaling environment, however, the role and regulation of ADAMs in renal fibrosis remain unclear. TGF-β stimulation of renal cells results in a significant up-regulation of Adams 10, 17, 12, and 19. The selective Smad2/3 inhibitor SB 525334 reversed these TGF-β–induced changes. In vivo, using ureteral obstruction to model renal fibrosis, we observed increased Adams gene expression that was blocked by oral administration of SB 525334. Similar increases in Adam gene expression also occurred in preclinical models of hypertension-induced renal damage and glomerulonephritis. miRNAs are a recently discovered second level of regulation of gene expression. Analysis of 3′ untranslated regions of Adam12 and Adam19 mRNAs showed multiple binding sites for miR-29a, miR-29b, and miR-29c. We show that miR-29 family expression is decreased after unilateral ureter obstruction and this significant decrease in miR-29 family expression was observed consistently in preclinical models of renal dysfunction and correlated with an increase in Adam12 and Adam19 expression. Exogenous overexpression of the miR-29 family blocked TGF-β–mediated up-regulation of Adam12 and Adam19 gene expression. This study shows that Adams are involved in renal fibrosis and are regulated by canonical TGF-β signaling and miR-29. Therefore, both Adams and the miR-29 family represent therapeutic targets for renal fibrosis.

diabetic nephropathy (DN) remains the single most common cause of end-stage kidney disease, necessitating dialysis or transplantation, in the Western world. Hence, novel therapies beyond tight blood pressure and glycemic control are required to slow or reverse progression of nephropathy in patients

Dysregulation of microRNAs (miRNAs) can contribute to the etiology of diseases, including pulmonary arterial hypertension (PAH). Here we investigated a potential role for the miR-214 stem loop miRNA and the closely linked miR-199a miRNAs in PAH. All 4 miRNAs were upregulated in the lung and right ventricle (RV) in mice and rats exposed to the Sugen (SU) 5416 hypoxia model of PAH. Further, expression of the miRNAs was increased in pulmonary artery smooth muscle cells exposed to transforming growth factor β1 but not BMP4. We then examined miR-214(-/-) mice exposed to the SU 5416 hypoxia model of PAH or normoxic conditions and littermate controls. There were no changes in RV systolic pressure or remodeling observed between the miR-214(-/-) and wild-type hypoxic groups. However, we observed a significant increase in RV hypertrophy (RVH) in hypoxic miR-214(-/-) male mice compared with controls. Further, we identified that the validated miR-214 target phosphatase and tensin homolog was upregulated in miR-214(-/-) mice. Thus, miR-214 stem loop loss leads to elevated RVH and may contribute to the heart failure associated with PAH.

A major limitation for adenoviral transduction in vivo is the profound liver tropism of adenovirus type 5 (Ad5). Recently, we demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity to mediate hepatocyte transduction after intravascular delivery. We developed novel genetically FX-binding ablated Ad5 vectors with lower liver transduction. Here, we demonstrate that FX-binding ablated Ad5 predominantly localize to the liver and spleen 1 hour after injection; however, they had highly reduced liver transduction in both control and macrophage-depleted mice compared with Ad5. At high doses in macrophage-depleted mice, FX-binding ablated vectors transduced the spleen more efficiently than Ad5. Immunohistochemical studies demonstrated transgene colocalization with CD11c+, ER-TR7+, and MAdCAM-1+ cells in the splenic marginal zone. Systemic inflammatory profiles were broadly similar between FX-binding ablated Ad5 and Ad5 at low and intermediate doses, although higher levels of several inflammatory proteins were observed at the highest dose of FX-binding ablated Ad5. Subsequently, we generated a FX-binding ablated virus containing a high affinity Ad35 fiber that mediated a significant improvement in lung/liver ratio in macrophage-depleted CD46+ mice compared with controls. Therefore, this study documents the biodistribution and reports the retargeting capacity of FX binding-ablated Ad5 vectors in vitro and in vivo.

Objective— MicroRNAs (miRNAs) are small noncoding RNAs that have the capacity to control protein production through binding “seed” sequences within a target mRNA. Each miRNA is capable of potentially controlling hundreds of genes. The regulation of miRNAs in the lung during the development of pulmonary arterial hypertension (PAH) is unknown. Methods and Results— We screened lung miRNA profiles in a longitudinal and crossover design during the development of PAH caused by chronic hypoxia or monocrotaline in rats. We identified reduced expression of Dicer, involved in miRNA processing, during the onset of PAH after hypoxia. MiR-22, miR-30, and let-7f were downregulated, whereas miR-322 and miR-451 were upregulated significantly during the development of PAH in both models. Differences were observed between monocrotaline and chronic hypoxia. For example, miR-21 and let-7a were significantly reduced only in monocrotaline-treated rats. MiRNAs that were significantly regulated were validated by quantitative polymerase chain reaction. By using in vitro studies, we demonstrated that hypoxia and growth factors implicated in PAH induced similar changes in miRNA expression. Furthermore, we confirmed miR-21 downregulation in human lung tissue and serum from patients with idiopathic PAH. Conclusion— Defined miRNAs are regulated during the development of PAH in rats. Therefore, miRNAs may contribute to the pathogenesis of PAH and represent a novel opportunity for therapeutic intervention.

Despite some advances, patients with advanced renal cell carcinoma (RCC) cannot usually be cured. Alteration of the natural tropism of adenoviruses may permit more specific gene transfer to target tissues. The aim of this study was to use novel targeting moieties for adenoviral gene therapy of RCC. Previous work in rats suggested that use of Ad5/19p (Ad5 capsid with Ad19p fiber) with kidney vascular targeting moieties HTTHREP (HTT), HITSLLS (HIT), and APASLYN (APA) placed into the fiber knob might be useful for targeting kidney vasculature. Therefore, we sought to investigate the utility of Ad5/19p variants for gene delivery to human RCC cell lines, clinical samples, and orthotopic murine models of metastatic RCC. Six different human RCC cell lines were infected but only Ad5/19p-HIT showed increased transduction, and only in one cell line. Thus, we analyzed human normal and cancerous kidney specimens fresh from patients, which might better mimic the three-dimensional architecture of clinical tumors and found that Ad5/19p-HIT showed transduction levels similar to Ad5. In mice, we found that intraperitoneal and intravenous Ad5/19p-HIT transduced tumors at levels comparable to Ad5, and that intratumoral Ad5/19p-HIT was superior to Ad5. Liver tropism was significantly reduced in comparison with Ad5. Improvements in tumor-to-liver transduction ratios suggested that Ad5/19p-HIT may be promising for systemic gene delivery to kidney tumors.

SummaryAdenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.

The potential efficacy of gene delivery is dictated by the infectivity profile of existing vectors, which is often restrictive. In order to target cells and organs for which no efficient vector is currently available, a promising approach would be to engineer vectors with novel transduction profiles. Applications that involve injecting adenovirus (Ad) vectors into the bloodstream require that native tropism for the liver be removed, and that targeting moieties be engineered into the capsid. We previously reported that pseudotyping the Ad serotype 5 fiber for that of Ad19p results in reduced hepatic transduction. In this study we show that this may be caused, at least in part, by a reduction in the capacity of the Ad19p-based virus to bind blood coagulation factors. It is therefore a potential candidate for vector retargeting, focusing on the kidney as a therapeutic target. We used in vivo phage display in rats, and identified peptides HTTHREP and HITSLLS that homed to the kidneys following intravenous injection. We engineered the HI loop of Ad19p to accommodate peptide insertions and clones. Intravenous delivery of each peptide-modified virus resulted in selective renal targeting, with HTTHREP and HITSLLS-targeted viruses selectively transducing tubular epithelium and glomeruli, respectively. Our study has important implications for the use of genetic engineering of Ad fibers to produce targeted gene delivery vectors.

MicroRNAs (miRNA) are small non-coding RNA molecules representing a novel class of endogenous negative regulators of gene expression. MiRNA have the ability to bind to specific regions in the 3'UTR of mRNA and repress gene expression through interaction with the RNA induced silencing complex (RISC). They have now been implicated in the pathophysiology of many kidney diseases, including the onset and progression of tubulointerstitial and glomerulosclerosis and have potential as biomarkers and as novel targets for treatment. The unique feature of miRNAs to target multiple mRNAs defines that targeting a particular miRNA for therapy could have a dramatic effect on the disease process. This review will focus on our current understanding of the role of miRNA in renal diseases, including diabetes, renal fibrosis, IgA nephropathy and explore the miRNA targets which represent the most promising in terms of clinical translation.

Adenovirus (Ad5) serotype 5 vectors are commonly used for gene transfer. Preclinical studies have shown that their application to systemic gene delivery, however, is limited by their highly efficient uptake in the liver, principally mediated by receptor-binding sites on the fiber shaft and knob domain. Using Ad to target other sites in vivo requires vectors that lack hepatic tropism. We therefore sought to exploit Ad family diversity to isolate vectors that possessed poor hepatic tropism. We pseudotyped the fibers from Ad16 (subgroup B; Ad5/16), Ad19p (subgroup D; Ad5/19p), and Ad37 (subgroup D; Ad5/37) onto Ad5 capsids and assessed infectivity profiles in vitro in multiple cell types and in vivo in rats. In rat, mouse, and human hepatocytes, Ad5/19p and Ad5/37 both possessed a striking lack of hepatic cell infectivity compared with Ad5. Both vectors were, however, able to transduce human vascular endothelial and smooth muscle cells with efficiencies equal to or greater than that of nonmodified Ad5. We evaluated liver uptake in 12-week-old male rats after intravenous injection. In contrast to a vector with the wild-type Ad5 fiber, Ad5, both Ad5/19p and Ad5/37 produced significantly less virion accumulation (measured at 1 hr and 5 days) and transgene expression in the liver. Thus, Ad5/19p and Ad5/37 may be useful platforms for the development of targeted Ad vectors.

Grafting of saphenous vein (SV) conduits into the arterial circulation triggers a number of adaptive pathological changes characterized by progressive medial thickening, neointima formation and accelerated atheroma. Previous studies have shown that modification of vein graft biology is possible by adenovirus (Ad)-mediated gene transfer, although gene expression is transient. Advancement of vascular gene therapy to the clinic is compromised by the lack of safe and efficient vector systems that provide sustained therapeutic gene delivery to the vasculature. Due to inadequacies of both Ad and adeno-associated virus (AAV) serotype-2 (AAV-2) systems, we have evaluated gene delivery to endothelial cells (ECs) and smooth muscle cells (SMCs) using alternate AAV serotypes and a third-generation vesicular stomatis virus glycoprotein-pseudotyped lentiviral system. Transduction of both primary human SV EC and SMC was lower using all alternate AAV serotypes compared to AAV-2. However, transduction of both cell types by lentivirus was efficient even at clinically relevant exposure times (15 min), was without toxicity and was promoter sensitive. Transduction levels at lower doses were further enhanced with the addition of the surfactant Poloxamer-407 (P-407). Direct comparison with Ad and AAV-2 confirmed the unique potential for this system. Moreover, we constructed and overexpressed the therapeutic gene tissue inhibitor of metalloproteinase-3 (TIMP-3) using lentivirus and demonstrated transgene production comparable to Ad with concomitant blockade of SMC migration and induction of cell death. We have demonstrated for the first time the potential for third-generation lentiviral vectors, but not alternate AAV serotypes, as efficient vascular gene delivery vectors.

The last decade has seen substantial advances in the development of gene therapy strategies and vector technology for the treatment of a diverse number of diseases, with a view to translating the successes observed in animal models into the clinic. Perhaps the overwhelming drive for the increase in vascular gene transfer studies is the current lack of successful long-term pharmacological treatments for complex cardiovascular diseases. The increase in cardiovascular disease to epidemic proportions has also led many to conclude that drug therapy may have reached a plateau in its efficacy and that gene therapy may represent a realistic solution to a long-term problem. Here, we discuss gene delivery approaches and target diseases.