Recent single-cell RNA sequencing (scRNA-seq) analyses have offered much insight into cell-specific gene expression profiles in normal kidneys. However, in diseased kidneys, understanding of changes ...in specific cells, particularly glomerular cells, remains limited.
To elucidate the glomerular cell-specific gene expression changes in diabetic kidney disease, we performed scRNA-seq analysis of isolated glomerular cells from streptozotocin-induced diabetic endothelial nitric oxide synthase (eNOS)-deficient (eNOS
) mice and control eNOS
mice.
We identified five distinct cell populations, including glomerular endothelial cells, mesangial cells, podocytes, immune cells, and tubular cells. Using scRNA-seq analysis, we confirmed the expression of glomerular cell-specific markers and also identified several new potential markers of glomerular cells. The number of immune cells was significantly higher in diabetic glomeruli compared with control glomeruli, and further cluster analysis showed that these immune cells were predominantly macrophages. Analysis of differential gene expression in endothelial and mesangial cells of diabetic and control mice showed dynamic changes in the pattern of expressed genes, many of which are known to be involved in diabetic kidney disease. Moreover, gene expression analysis showed variable responses of individual cells to diabetic injury.
Our findings demonstrate the ability of scRNA-seq analysis in isolated glomerular cells from diabetic and control mice to reveal dynamic changes in gene expression in diabetic kidneys, with variable responses of individual cells. Such changes, which might not be apparent in bulk transcriptomic analysis of glomerular cells, may help identify important pathophysiologic factors contributing to the progression of diabetic kidney disease.
To identify the factors mediating the progression of diabetic nephropathy (DN), we performed RNA sequencing of kidney biopsy samples from patients with early DN, advanced DN, and normal kidney tissue ...from nephrectomy samples. A set of genes that were upregulated at early but downregulated in late DN were shown to be largely renoprotective, which included genes in the retinoic acid pathway and glucagon-like peptide 1 receptor. Another group of genes that were downregulated at early but highly upregulated in advanced DN consisted mostly of genes associated with kidney disease pathogenesis, such as those related to immune response and fibrosis. Correlation with estimated glomerular filtration rate (eGFR) identified genes in the pathways of iron transport and cell differentiation to be positively associated with eGFR, while those in the immune response and fibrosis pathways were negatively associated. Correlation with various histopathological features also identified the association with the distinct gene ontological pathways. Deconvolution analysis of the RNA sequencing data set indicated a significant increase in monocytes, fibroblasts, and myofibroblasts in advanced DN kidneys. Our study thus provides potential molecular mechanisms for DN progression and association of differential gene expression with the functional and structural changes observed in patients with early and advanced DN.
Renal inflammation and fibrosis are the common pathways leading to progressive chronic kidney disease (CKD). We previously identified hematopoietic cell kinase (HCK) as upregulated in human chronic ...allograft injury promoting kidney fibrosis; however, the cellular source and molecular mechanisms are unclear. Here, using immunostaining and single cell sequencing data, we show that HCK expression is highly enriched in pro-inflammatory macrophages in diseased kidneys. HCK-knockout (KO) or HCK-inhibitor decreases macrophage M1-like pro-inflammatory polarization, proliferation, and migration in RAW264.7 cells and bone marrow-derived macrophages (BMDM). We identify an interaction between HCK and ATG2A and CBL, two autophagy-related proteins, inhibiting autophagy flux in macrophages. In vivo, both global or myeloid cell specific HCK-KO attenuates renal inflammation and fibrosis with reduces macrophage numbers, pro-inflammatory polarization and migration into unilateral ureteral obstruction (UUO) kidneys and unilateral ischemia reperfusion injury (IRI) models. Finally, we developed a selective boron containing HCK inhibitor which can reduce macrophage pro-inflammatory activity, proliferation, and migration in vitro, and attenuate kidney fibrosis in the UUO mice. The current study elucidates mechanisms downstream of HCK regulating macrophage activation and polarization via autophagy in CKD and identifies that selective HCK inhibitors could be potentially developed as a new therapy for renal fibrosis.
Interstitial fibrosis, tubular atrophy, and inflammation are major contributors to kidney allograft failure. Here we sought an objective, quantitative pathological assessment of these lesions to ...improve predictive utility and constructed a deep-learning–based pipeline recognizing normal vs. abnormal kidney tissue compartments and mononuclear leukocyte infiltrates. Periodic acid– Schiff stained slides of transplant biopsies (60 training and 33 testing) were used to quantify pathological lesions specific for interstitium, tubules and mononuclear leukocyte infiltration. The pipeline was applied to the whole slide images from 789 transplant biopsies (478 baseline pre-implantation and 311 post-transplant 12-month protocol biopsies) in two independent cohorts (GoCAR: 404 patients, AUSCAD: 212 patients) of transplant recipients to correlate composite lesion features with graft loss. Our model accurately recognized kidney tissue compartments and mononuclear leukocytes. The digital features significantly correlated with revised Banff 2007 scores but were more sensitive to subtle pathological changes below the thresholds in the Banff scores. The Interstitial and Tubular Abnormality Score (ITAS) in baseline samples was highly predictive of one-year graft loss, while a Composite Damage Score in 12-month post-transplant protocol biopsies predicted later graft loss. ITASs and Composite Damage Scores outperformed Banff scores or clinical predictors with superior graft loss prediction accuracy. High/intermediate risk groups stratified by ITASs or Composite Damage Scores also demonstrated significantly higher incidence of estimated glomerular filtration rate decline and subsequent graft damage. Thus, our deep-learning approach accurately detected and quantified pathological lesions from baseline or post-transplant biopsies and demonstrated superior ability for prediction of post-transplant graft loss with potential application as a prevention, risk stratification or monitoring tool.
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Abstract
Background
Long Interspersed Nuclear Element-1 (LINE-1, L1) is increasingly regarded as a genetic risk for lung cancer. Transcriptionally active LINE-1 forms a L1-gene chimeric transcript ...(LCTs), through somatic L1 retrotransposition (LRT) or L1 antisense promoter (L1-ASP) activation, to play an oncogenic role in cancer progression.
Methods
Here, we developed Retrotransposon-gene fusion estimation program (ReFuse), to identify and quantify LCTs in RNA sequencing data from TCGA lung cancer cohort (
n
= 1146) and a single cell RNA sequencing dataset then further validated those LCTs in an independent cohort (
n
= 134). We next examined the functional roles of a cancer specific LCT (
L1-FGGY
) in cell proliferation and tumor progression in LUSC cell lines and mice.
Results
The LCT events correspond with specific metabolic processes and mitochondrial functions and was associated with genomic instability, hypomethylation, tumor stage and tumor immune microenvironment (TIME). Functional analysis of a tumor specific and frequent LCT involving
FGGY
(
L1-FGGY
) reveal that the arachidonic acid (AA) metabolic pathway was activated by the loss of
FGGY
through the
L1-FGGY
chimeric transcript to promote tumor growth, which was effectively targeted by a combined use of an anti-HIV drug (NVR) and a metabolic inhibitor (ML355). Lastly, we identified a set of transcriptomic signatures to stratify the LUSC patients with a higher risk for poor outcomes who may benefit from treatments using NVR alone or combined with an anti-metabolism drug.
Conclusions
This study is the first to characterize the role of L1 in metabolic reprogramming of lung cancer and provide rationale for L1-specifc prognosis and potential for a therapeutic strategy for treating lung cancer.
Trial registration
Study on the mechanisms of the mobile element
L1-FGGY
promoting the proliferation, invasion and immune escape of lung squamous cell carcinoma through the 12-LOX/Wnt pathway, Ek2020111. Registered 27 March 2020 ‐ Retrospectively registered.
Donor-recipient (D-R) mismatches outside of human leukocyte antigens (HLAs) contribute to kidney allograft loss, but the mechanisms remain unclear, specifically for intronic mismatches. We quantified ...non-HLA mismatches at variant-, gene-, and genome-wide scales from single nucleotide polymorphism (SNP) data of D-Rs from 2 well-phenotyped transplant cohorts: Genomics of Chronic Allograft Rejection (GoCAR; n = 385) and Clinical Trials in Organ Transplantation-01/17 (CTOT-01/17; n = 146). Unbiased gene-level screening in GoCAR uncovered the LIMS1 locus as the top-ranked gene where D-R mismatches associated with death-censored graft loss (DCGL). A previously unreported, intronic, LIMS1 haplotype of 30 SNPs independently associated with DCGL in both cohorts. Haplotype mismatches showed a dosage effect, and minor-allele introduction to major-allele-carrying recipients showed greater hazard of DCGL. The LIMS1 haplotype and the previously reported LIMS1 SNP rs893403 are expression quantitative trait loci (eQTL) in immune cells for GCC2 (not LIMS1), which encodes a protein involved in mannose-6-phosphase receptor (M6PR) recycling. Peripheral blood and T cell transcriptome analyses associated the GCC2 gene and LIMS1 SNPs with the TGF-β1/SMAD pathway, suggesting a regulatory effect. In vitro GCC2 modulation impacted M6PR-dependent regulation of active TGF-β1 and downstream signaling in T cells. Together, our data link LIMS1 locus D-R mismatches to DCGL via GCC2 eQTLs that modulate TGF-β1-dependent effects on T cells.
Well characterized the connections among diseases, long non-coding RNAs (lncRNAs) and drugs are important for elucidating the key roles of lncRNAs in biological mechanisms in various biological ...states. In this study, we constructed a database called LNCmap (LncRNA Connectivity Map), available at http://www.bio-bigdata.com/LNCmap/ , to establish the correlations among diseases, physiological processes, and the action of small molecule therapeutics by attempting to describe all biological states in terms of lncRNA signatures. By reannotating the microarray data from the Connectivity Map database, the LNCmap obtained 237 lncRNA signatures of 5916 instances corresponding to 1262 small molecular drugs. We provided a user-friendly interface for the convenient browsing, retrieval and download of the database, including detailed information and the associations of drugs and corresponding affected lncRNAs. Additionally, we developed two enrichment analysis methods for users to identify candidate drugs for a particular disease by inputting the corresponding lncRNA expression profiles or an associated lncRNA list and then comparing them to the lncRNA signatures in our database. Overall, LNCmap could significantly improve our understanding of the biological roles of lncRNAs and provide a unique resource to reveal the connections among drugs, lncRNAs and diseases.
Cholesterol 25‐hydroxylase (CH25H), an enzyme involved in cholesterol metabolism, regulates inflammatory responses and lipid metabolism. However, its role in kidney disease is not known. The author ...found that CH25H transcript is expressed mostly in glomerular and peritubular endothelial cells and that its expression increased in human and mouse diabetic kidneys. Global deletion of Ch25h in Leprdb/db mice aggravated diabetic kidney disease (DKD), which is associated with increased endothelial cell apoptosis. Treatment of 25‐hydroxycholesterol (25‐HC), the product of CH25H, alleviated kidney injury in Leprdb/db mice. Mechanistically, 25‐HC binds to GTP‐binding protein ADP‐ribosylation factor 4 (ARF4), an essential protein required for maintaining protein transport in the Golgi apparatus. Interestingly, ARF4's GTPase‐activating protein ASAP1 is also predominantly expressed in endothelial cells and its expression increased in DKD. Suppression of ARF4 activity by deleting ARF4 or overexpressing ASAP1 results in endothelial cell death. These results indicate that 25‐HC binds ARF4 to inhibit its interaction with ASAP1, and thereby resulting in enhanced ARF4 activity to confer renoprotection. Therefore, treatment of 25‐HC improves kidney injury in DKD in part by restoring ARF4 activity to maintain endothelial cell survival. This study provides a novel mechanism and a potential new therapy for DKD.
This study shows that cholesterol 25‐hydroxylase (CH25H), a key enzyme in cholesterol metabolism, is expressed in kidney endothelial cells and protects against diabetic kidney disease. Its product, 25‐HC, binds to and enhances the activity of GTP‐binding protein ADP‐ribosylation factor 4 (ARF4), an essential protein required for Golgi trafficking and homeostasis, to promote endothelial cell survival and slow disease progression.
Anemia commonly occurs in systemic lupus erythematosus (SLE), a disease characterized by innate immune activation by nucleic acids. Overactivation of cytoplasmic sensors by self-DNA or RNA can cause ...erythroid cell death while sparing other hematopoietic cell lineages. However, little is currently known about the impact of nucleic acid sensing innate receptors on the bone marrow (BM) erythropoietic niche.The ssRNA endosomal receptors TLR7 and human TLR8 both recognize ssRNA. TLR7 overexpression causes a lupus syndrome that includes the development of mild to moderate anemia (Hb >10) and thrombocytopenia and is associated with spleen histiocytosis, autoimmune hemolysis, erythrophagocytosis and compensatory stress erythropoiesis in the spleen. A recent study demonstrated that patients with TLR8 gain of function present with immunodeficiency, inflammation and bone marrow failure. However, the role of TLR8 in SLE has been difficult to study in mice because it has a 5 amino acid deletion that attenuates its RNA binding capacity.To address the role of TLR8 in SLE, we overexpressed human TLR8 in a lupus mouse model (huTLR8tg.Sle1.Yaa) using a BAC transgene. 50% of homozygous huTLR8tg.Sle1.Yaa mice developed severe anemia (Hb < 9) resulting in early mortality starting at 3–4.5 months of age. This phenotype required both the Sle1 and Yaa loci that promote the formation of high titer anti-chromatin and anti-RNA antibodies and onset of nephritis at > 6 months of age. There was no difference in autoantibody titers between Sle1.Yaa wt and huTLR8tg mice and early death in the transgenic mice was not due to premature onset of renal disease. All mice had normal RBC indices prior to 10 weeks of age. Anemia was associated with an increase in bone marrow (BM) trabecular bone and a decrease in erythromyeloblastic islands (EMBI) in the BM with compensatory stress erythropoiesis leading to reticulocytosis and vast splenomegaly. RBC half- life decreased in severely anemic mice as a pre-terminal event and was due to hemophagocytosis by 3 subsets of phagocytic red pulp macrophages.Flow cytometry of BMs showed a block in CFU-E to proerythroblast differentiation that was confirmed by single cell RNASeq of bone marrow EMBIs. The erythroblast cluster proximal to the block had a signature of mitochondrial stress and decreased proliferation. We found 6 closely related clusters of EMBI central macrophages in the BM, most of which displayed an inflammatory and Type 1 IFN signature. One cluster (M2) expressed all the classical central macrophage phenotypic markers was characterized in transgenic mice by downregulation of multiple phagocytic receptors and a 5-fold decrease of VCAM1 expression. Loss of VCAM1 and downregulation of Mertk and CD169 in BM central macrophages was confirmed by flow cytometry. By contrast spleen central macrophages from transgenic mice retained VCAM1 and CD169 expression (figures 1–3).Together, these results suggest that erythropoiesis in the BM of huTLR8tg SLE-prone mice fails due to a block in differentiation from CFU-E to proerythroblasts in the BM and is associated with an inflammatory phenotype specifically in BM erythroblastic island central macrophages and down regulation of adhesion and phagocytic receptors. These data implicate the endosomal RNA sensor TLR8 as an additional innate receptor whose overactivation causes acquired failure of erythropoiesis via myeloid cell dysregulation. Compensatory stress erythropoiesis in the spleens is associated with expansion of several subsets of macrophages with phagocytic properties and fatal anemia is associated with a decrease in red blood cell half-life, suggesting that excessive RBC phagocytosis, coupled with insufficient erythroblast progenitors, eventually exceeds the capability of stress erythropoiesis to replace the RBC mass.Abstract 801 Figure 1Premature death in huTLR8tg Sle1.Yaa mice is due to severe anemia that is due to loss of RBC precursors at the proerythroblast stage as shown by flow cytometry of bone marrowAbstract 801 Figure 2Single cell analysis of isolated BM EMBIs shows a decrease in central macrophage adhesion molecules, confirmed by flow cytometryAbstract 801 Figure 3Decrease in RBC half-life in anemic mice (A) is acquired when RBC from young mice are transferred to anemic mice (B). Hemophagocytic macrophages (C) from anemic mice comprise 3 subsets including iHPCs and red pulp macrophages (D).
Podocyte injury and loss are key drivers of primary and secondary glomerular diseases, such as focal segmental glomerulosclerosis (FSGS) and diabetic kidney disease (DKD). We previously demonstrated ...the renoprotective role of protein S (PS) and its cognate tyrosine-protein kinase receptor, TYRO3, in models of FSGS and DKD and that their signaling exerts antiapoptotic and antiinflammatory effects to confer protection against podocyte loss. Among the 3 TAM receptors (TYRO3, AXL, and MER), only TYRO3 expression is largely restricted to podocytes, and glomerular TYRO3 mRNA expression negatively correlates with human glomerular disease progression. Therefore, we posited that the agonistic PS/TYRO3 signaling could serve as a potential therapeutic approach to attenuate glomerular disease progression. As PS function is not limited to TYRO3-mediated signal transduction but includes its anticoagulant activity, we focused on the development of TYRO3 agonists as an optimal therapeutic approach to glomerular disease. Among the small-molecule TYRO3 agonistic compounds screened, compound 10 (C-10) showed a selective activation of TYRO3 without any effects on AXL or MER. We also confirmed that C-10 directly binds to TYRO3, but not the other receptors. In vivo, C-10 attenuated proteinuria, glomerular injury, and podocyte loss in mouse models of Adriamycin-induced nephropathy and a db/db model of type 2 diabetes. Moreover, these renoprotective effects of C-10 were lost in Tyro3-knockout mice, indicating that C-10 is a selective agonist of TYRO3 activity that mitigates podocyte injury and glomerular disease. Therefore, C-10, a TYRO3 agonist, could be potentially developed as a new therapy for glomerular disease.
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