Kidney Network

Kidney Network - Overview

Aa comprehensive scRNA-seq analysis of human renal biopsies from IgAN. We showed for the first time that IgAN mesangial cells displayed increased expression of several novel genes including MALAT1, GADD45B, SOX4, and EDIL3, which were related to cell proliferation and matrix accumulation. The overexpressed genes in tubule cells of IgAN were mainly enriched in inflammatory pathways including TNF signaling, IL-17 signaling, and NOD-like receptor signaling. Furthermore, we compared the results of 4 IgAN patients with the published scRNA-Seq data of healthy kidney tissues of three human donors in order to further validate the findings in our study. The results also verified that the overexpressed genes in tubule cells from IgAN patients were mainly enriched in inflammatory pathways including TNF signaling, IL-17 signaling, and NOD-like receptor signaling. The receptor-ligand crosstalk analysis revealed potential interactions between mesangial cells and other cells in IgAN. IgAN patients with overt proteinuria displayed elevated genes participating in several signaling pathways compared with microproteinuria group. It needs to be mentioned that based on number of mesangial cells and other kidney cells analyzed in this study, the results of our study are preliminary and needs to be confirmed on larger number of cells from larger number of patients and controls in future studies. Therefore, these results offer new insight into pathogenesis and identify new therapeutic targets for IgAN.

A Partial Picture of the Single-Cell Transcriptomics of Human IgA Nephropathy

The gene expression program underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of gene expression and chromatin accessibility in fetal tissues. For gene expression, we applied three-level combinatorial indexing to >110 samples representing 15 organs, ultimately profiling ~4 million single cells. We leveraged the literature and other atlases to identify and annotate hundreds of cell types and subtypes, both within and across tissues. Our analyses focused on organ-specific specializations of broadly distributed cell types (such as blood, endothelial, and epithelial), sites of fetal erythropoiesis (which notably included the adrenal gland), and integration with mouse developmental atlases (such as conserved specification of blood cells). These data represent a rich resource for the exploration of in vivo human gene expression in diverse tissues and cell types.

A human cell atlas of fetal gene expression.

Single-cell RNA sequencing (scRNA-seq) is the leading technique for charting the molecular properties of individual cells. The latest methods are scalable to thousands of cells, enabling in- depth characterization of sample composition without prior knowledge. However, there are important differences between scRNA-seq techniques, and it remains unclear which are the most suitable protocols for drawing cell atlases of tissues, organs and organisms. We have generated benchmark datasets to systematically evaluate techniques in terms of their power to comprehensively describe cell types and states. We performed a multi-center study comparing 13 commonly used single-cell and single-nucleus RNA-seq protocols using a highly heterogeneous reference sample resource. Comparative and integrative analysis at cell type and state level revealed marked differences in protocol performance, highlighting a series of key features for cell atlas projects. These should be considered when defining guidelines and standards for international consortia, such as the Human Cell Atlas project. Overall design: The samples consists of two complex tissues (human PBMC and mouse colon) and three cell lines (HEK293-RFP, NIH3T3-GFP, MDCK-Turbo650). The primary PBMC and the colon sample constitute 90% and the cell lines 10%. The sample preparation aims to be standardized for all methods to allow a comparison of library preparation performance (6% HEK293T-RFP, 3% NIH3T3-GFP, 1% MDCK-Turbo650) of the sample content.

Benchmarking Single-Cell RNA Sequencing Protocols for Cell Atlas Projects

The authors generated single-cell atlases of 23 lung, 16 kidney, 15 liver and 18 heart COVID-19 autopsy donor tissue samples, and spatial atlases of 14 lung donors.

COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets.

We analyzed single cell transcriptomes over 80,000 cells isolated from 65 organoids differentiated from iPSCs and ESCs using two different protocols. We find that both protocols generate kidney organoids that contain a diverse range of kidney cells at differing ratios as well as non-renal cell types. We reconstructed lineage relationships during organoid differentiation through pseudotemporal ordering, and identified transcription factor networks associated with fate decisions. When comparing to adult human kidney, we reveal immaturity of all kidney organoid cell types. These results define impressive kidney organoid cell diversity, identify incomplete differentiation as a major roadblock for current directed differentiation protocols and provide a human adult kidney snRNA-seq dataset against which to benchmark future progress. Overall design: Dropseq and 10X Chromium were used to profile organoid cells and adult kidney nuclei

Comparative Analysis and Refinement of Human PSC-Derived Kidney Organoid Differentiation with Single-Cell Transcriptomics

Single-cell RNA sequencing was used to determine the cell type composition of all major human organs and construct a basic scheme for the human cell landscape (HCL).

Construction of A Human Cell Landscape by Single-cell mRNA-seq

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells/multipotent progenitors (HSC/MPPs), yet remains poorly defined in humans. Using single cell transcriptome profiling of ~133,000 fetal liver and ~65,000 fetal skin and kidney cells, we identify the repertoire of blood and immune cells in first and early second trimesters of development. From this data, we infer differentiation trajectories from HSC/MPPs, and evaluate the impact of tissue microenvironment on blood and immune cell development. We predict coupling of mast cell differentiation with erythro-megakaryopoiesis and identify physiological erythropoiesis in fetal skin. We demonstrate a shift in fetal liver haematopoietic composition during gestation away from being erythroid-predominant, accompanied by a parallel change in HSC/MPP differentiation potential, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a valuable reference for understanding and harnessing the therapeutic potential of HSC/MPPs.

Decoding human fetal liver haematopoiesis

Autosomal dominant polycystic kidney disease (ADPKD) is the leading genetic cause of end stage renal disease characterized by progressive expansion of kidney cysts. To better understand the cell types and states driving ADPKD progression, we analyze eight ADPKD and five healthy human kidney samples, generating single cell multiomic atlas consisting of ~100,000 single nucleus transcriptomes and ~50,000 single nucleus epigenomes. Activation of proinflammatory, profibrotic signaling pathways are driven by proximal tubular cells with a failed repair transcriptomic signature, proinflammatory fibroblasts and collecting duct cells. We identify GPRC5A as a marker for cyst-lining collecting duct cells that exhibits increased transcription factor binding motif availability for NF-κB, TEAD, CREB and retinoic acid receptors. We identify and validate a distal enhancer regulating GPRC5A expression containing these motifs. This single cell multiomic analysis of human ADPKD reveals previously unrecognized cellular heterogeneity and provides a foundation to develop better diagnostic and therapeutic approaches.

Defining cellular complexity in human autosomal dominant polycystic kidney disease by multimodal single cell analysis

We tested the hypothesis that single-cell RNA-sequencing (scRNA-seq) analysis of human kidney allograft biopsies will reveal distinct cell types and states and yield insights to decipher the complex heterogeneity of alloimmune injury. We selected 3 biopsies of kidney cortex from 3 individuals for scRNA-seq and processed them fresh using an identical protocol on the 10x Chromium platform; (i) HK: native kidney biopsy from a living donor, (ii) AK1: allograft kidney with transplant glomerulopathy, tubulointerstitial fibrosis, and worsening graft function, and (iii) AK2: allograft kidney after successful treatment of active antibody-mediated rejection. We did not study T-cell-mediated rejections. We generated 7217 high-quality single cell transcriptomes. Taking advantage of the recipient-donor sex mismatches revealed by X and Y chromosome autosomal gene expression, we determined that in AK1 with fibrosis, 42 months after transplantation, more than half of the kidney allograft fibroblasts were recipient-derived and therefore likely migratory and graft infiltrative, whereas in AK2 without fibrosis, 84 months after transplantation, most fibroblasts were donor-organ-derived. Furthermore, AK1 was enriched for tubular progenitor cells overexpressing profibrotic extracellular matrix genes. AK2, eight months after successful treatment of rejection, contained plasmablast cells with high expression of immunoglobulins, endothelial cell elaboration of T cell chemoattractant cytokines, and persistent presence of cytotoxic T cells. In addition to these key findings, our analysis revealed unique cell types and states in the kidney. Altogether, single-cell transcriptomics yielded novel mechanistic insights, which could pave the way for individualizing the care of transplant recipients.

Detection of infiltrating fibroblasts by single-cell transcriptomics in human kidney allografts

Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) lack some cellular populations found in the native organ, including vasculature. Using single-cell RNA sequencing (scRNA-seq), we have identified a population of endothelial cells (ECs) present early in HIO differentiation that declines over time in culture. Here, we developed a method to expand and maintain this endogenous population of ECs within HIOs (vHIOs). Given that ECs possess organ-specific gene expression, morphology, and function, we used bulk RNA-seq and scRNA-seq to interrogate the developing human intestine, lung, and kidney in order to identify organ-enriched EC gene signatures. By comparing these gene signatures and validated markers to HIO ECs, we find that HIO ECs grown in vitro share the highest similarity with native intestinal ECs relative to kidney and lung. Together, these data demonstrate that HIOs can co-differentiate a native EC population that is properly patterned with an intestine-specific EC transcriptional signature in vitro.

Differentiation of Human Intestinal Organoids with Endogenous Vascular Endothelial Cells

To investigate the gene expression profiles of human fetal kidney development, Wang et al. performed single-cell RNA-seq for >3,000 renal cells. They highlight the heterogeneity of cap mesenchyme and regulatory factors in nephron formation. The results reveal cell-type-specific risk genes in congenital renal diseases.

Dissecting the Global Dynamic Molecular Profiles of Human Fetal Kidney Development by Single-Cell RNA Sequencing

The datasets contain single cell RNA sequencing data from the normal region of tumor-nephrectomy samples.

HCA seed network precise tumor-nephrectomy samples

BackgroundIn solid organ transplantation, donor-derived immune cells are assumed to decline with time after surgery. Whether donor leukocytes persist within kidney transplants or play any role in rejection is unknown, however, in part because of limited techniques for distinguishing recipient from donor cells.MethodsWhole-exome sequencing of donor and recipient DNA and single-cell RNA sequencing (scRNA-seq) of five human kidney transplant biopsy cores distinguished immune cell contributions from both participants. DNA-sequence comparisons used single nucleotide variants (SNVs) identified in the exome sequences across all samples.ResultsAnalysis of expressed SNVs in the scRNA-seq data set distinguished recipient versus donor origin for all 81,139 cells examined. The leukocyte donor/recipient ratio varied with rejection status for macrophages and with time post-transplant for lymphocytes. Recipient macrophages displayed inflammatory activation whereas donor macrophages demonstrated antigen presentation and complement signaling. Recipient-origin T cells expressed cytotoxic and proinflammatory genes consistent with an effector cell phenotype, whereas donor-origin T cells appeared quiescent, expressing oxidative phosphorylation genes. Finally, both donor and recipient T cell clones within the rejecting kidney suggested lymphoid aggregation. The results indicate that donor-origin macrophages and T cells have distinct transcriptional profiles compared with their recipient counterparts, and that donor macrophages can persist for years post-transplantation.ConclusionsAnalysis of single nucleotide variants and their expression in single cells provides a powerful novel approach to accurately define leukocyte chimerism in a complex organ such as a transplanted kidney, coupled with the ability to examine transcriptional profiles at single-cell resolution.

Harnessing Expressed Single Nucleotide Variation and Single Cell RNA Sequencing To Define Immune Cell Chimerism in the Rejecting Kidney Transplant

The datasets contain single cell RNA sequencing data from healthy human kidneys. Samples were collected from healthy humans who were donating the kidney for the purpose of transplantation. Biopsies to obtain the samples were performed before the kidney was placed in the recipient to obtain cell states representative of the healthy donor.

Healthy human kidney cell type single cell RNA-seq data

To date, the pathogenesis of hepatitis B virus (HBV)-associated membranous nephropathy (MN) remains elusive. This study aimed to decipher the etiopathogenesis of HBV-associated MN by performing single-cell RNA sequencing (scRNA-seq) of kidney biopsy specimens from a patient with HBV-associated MN and two healthy individuals. We generated 4,114 intrarenal single-cell transcriptomes from the HBV-associated MN patient by scRNA-seq. Compared to healthy individuals, podocytes in the HBV-associated MN patient showed an increased expression of extracellular matrix formation-related genes, including HSPA5, CTGF, and EDIL3. Kidney endothelial cells (ECs) in the HBV-associated MN were enriched in inflammatory pathways, including NF-kappa B signaling, IL-17 signaling, TNF signaling and NOD-like receptor signaling. Gene ontology (GO) functional enrichment analysis and Gene Set Variation Analysis (GSVA) further revealed that differentially expressed genes (DEGs) of ECs from the HBV-associated MN patients were enriched in apoptotic signaling pathway, response to cytokine and leukocyte cell-cell adhesion. The up-regulated DEGs in glomerular ECs of HBV-associated MN patients were involved in biological processes such as viral gene expression, and protein targeting to endoplasmic reticulum. We further verified that the overexpressed genes in ECs from HBV-associated MN were mainly enriched in regulation of protein targeting to endoplasmic reticulum, exocytosis, viral gene expression, IL-6 and IL-1 secretion when compared with anti-phospholipase A2 receptor (PLA2R)-positive idiopathic membranous nephropathy (IMN). The receptor-ligand crosstalk analysis revealed potential interactions between endothelial cells and other cells in HBV-associated-MN. These results offer new insight into the pathogenesis of HBV-associated MN and may identify new therapeutic targets for HBV-associated MN.

Intrarenal Single-Cell Sequencing of Hepatitis B Virus Associated Membranous Nephropathy.

Here we describe sci-CAR, a combinatorial indexing strategy to jointly profile chromatin accessibility and mRNA in each of thousands of single cells. As a proof-of-concept, we apply sci-CAR to 4,825 cells comprising a time-series of dexamethasone treatment, as well as to 11,233 cells from the mouse kidney.

Joint profiling of chromatin accessibility and gene expression in thousands of single cells

Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8+ T cell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target.

Mapping single-cell transcriptomes in the intra-tumoral and associated territories of kidney cancer.

Recent advances in single cell genomics technologies have facilitated studies on the developing immune system at unprecedented scale and resolution. However, these studies have focused on one or a few organs and were thus limited in understanding the developing immune system as a distributed network across tissues. Here, we profiled prenatal haematopoietic organs, lymphoid organs and non-lymphoid tissues using a combination of single-cell RNA sequencing, paired antigen-receptor sequencing and spatial transcriptomics to reconstruct the developing human immune system. Our analysis revealed the acquisition of immune effector transcriptome profiles in macrophages, mast cells and NK cells from the second trimester, and the transcriptomic changes accompanying the late-stage maturation of developing monocytes and T cells that extended from their organ of origin to peripheral tissues. We uncovered system-wide blood and immune cell development beyond the conventional primary haematopoietic organs. We further identified, extensively characterised and functionally validated the human prenatal B1 cells. Finally, we provide evidence for thymocyte-thymocyte selection origin for αβTCR- expressing unconventional T cells based on TCR gene usage and an in vitro artificial thymic organoid culture model. Our comprehensive atlas of the developing human immune system provides both valuable data resources and biological insights that will facilitate cell engineering, regenerative medicine and disease understanding. 

One-Sentence Summary 
By performing a comprehensive single-cell RNA sequencing atlas of human developing immune system together with antigen-receptor sequencing and spatial transcriptomics, we explored the cross-gestation and cross-organ variability in immune cells, discovered system-wide blood and immune cell development, identified, characterised and functionally validated the properties of human prenatal B1 cells and the origin of unconventional T cells.

Mapping the developing human immune system across organs

In order to investigate heterogeneity and dynamics of cancer-associated immune cells, we performed single-cell sequencing on peripheral and tumor-infiltrating immune cells in three renal clear cell carcinoma patients. We chose renal clear cell carcinoma tumors based on the responsive of these tumors to immune checkpoint blockade in the context of low mutational loads, which implies a strong influence from the tumor microenvironment. Using the 10x Genomic 5 expression platform a total of 25,672 immune cells were isolated and passed filtering for quality control, with 13,433 cells from peripheral blood and 12,239 tumor-infiltrating cells. In addition, we used the Chromium Single Cell V(D)J kit to enrich for T cell receptor sequences for nearly 10,000 T cells in with accompanying expression information, allowing for the investigation of clonality and transcriptional phenotypic diversity. We generated a comprehensive immune profile using single-cell RNA-seq data from 25,000 immune cells from three renal cell carcinoma along with matched peripheral blood. In addition, we performed VDJ sequencing on the isolated single T cells. Overall design: T cell receptor sequences of the subset of T cells in the 5' RNA expression sequencing dataset.

Mapping the immune environment in clear cell renal carcinoma by single-cell genomics

Multimodal single cell sequencing is a powerful tool for interrogating cell-specific changes in transcription and chromatin accessibility. We performed single nucleus RNA (snRNA-seq) and assay for transposase accessible chromatin sequencing (snATAC-seq) on human kidney cortex from donors with and without diabetic kidney disease (DKD) to identify altered signaling pathways and transcription factors associated with DKD. Both snRNA-seq and snATAC-seq had an increased proportion of VCAM1+ injured proximal tubule cells (PT_VCAM1) in DKD samples. PT_VCAM1 has a pro-inflammatory expression signature and transcription factor motif enrichment implicated NFkB signaling. We used stratified linkage disequilibrium score regression to partition heritability of kidney-function-related traits using publicly-available GWAS summary statistics. Cell-specific PT_VCAM1 peaks were enriched for heritability of chronic kidney disease (CKD), suggesting that genetic background may regulate chromatin accessibility and DKD progression. snATAC-seq found cell-specific differentially accessible regions (DAR) throughout the nephron that change accessibility in DKD and these regions were enriched for glucocorticoid receptor (GR) motifs. Changes in chromatin accessibility were associated with decreased expression of insulin receptor, increased gluconeogenesis, and decreased expression of the GR cytosolic chaperone, FKBP5, in the diabetic proximal tubule. Cleavage under targets and release using nuclease (CUT&RUN) profiling of GR binding in bulk kidney cortex and an in vitro model of the proximal tubule (RPTEC) showed that DAR co-localize with GR binding sites. CRISPRi silencing of GR response elements (GRE) in the FKBP5 gene body reduced FKBP5 expression in RPTEC, suggesting that reduced FKBP5 chromatin accessibility in DKD may alter cellular response to GR. We developed an open-source tool for single cell allele specific analysis (SALSA) to model the effect of genetic background on gene expression. Heterozygous germline single nucleotide variants (SNV) in proximal tubule ATAC peaks were associated with allele-specific chromatin accessibility and differential expression of target genes within cis-coaccessibility networks. Partitioned heritability of proximal tubule ATAC peaks with a predicted allele-specific effect was enriched for eGFR, suggesting that genetic background may modify DKD progression in a cell-specific manner.

Multimodal single cell sequencing of human diabetic kidney disease implicates chromatin accessibility and genetic background in disease progression

Although it is well established that microbial infections predispose to autoimmune diseases, the underlying mechanisms remain poorly understood. After infection, tissue-resident memory T (TRM) cells persist in peripheral organs and provide immune protection against reinfection. However, whether TRM cells participate in responses unrelated to the primary infection, such as autoimmune inflammation, is unknown. By using high-dimensional single-cell analysis, we identified CD4+ TRM cells with a TH17 signature (termed TRM17 cells) in kidneys of patients with ANCA-associated glomerulonephritis. Experimental models demonstrated that renal TRM17 cells were induced by pathogens infecting the kidney, such as Staphylococcus aureus, Candida albicans, and uropathogenic Escherichia coli, and persisted after the clearance of infections. Upon induction of experimental glomerulonephritis, these kidney TRM17 cells rapidly responded to local proinflammatory cytokines by producing IL-17A and thereby exacerbate renal pathology. Thus, our data show that pathogen-induced TRM17 cells have a previously unrecognized function in aggravating autoimmune disease.

Pathogen-induced tissue-resident memory T <sub>H</sub> 17 (T <sub>RM</sub> 17) cells amplify autoimmune kidney disease

To characterize the cellular diversity in the human kidney cortical nephrogenic niche we dissociated cells from the cortex and performed 10X Genomics single-cell RNA sequencing.

Single Cell RNA-Seq profiling human embryonic kidney cortex cells

To study the developmental process of human podocytes and compare to the in vitro counterpart, we dissociated cells from the inner and outer kidney cortex as well as kidney organoids, and performed 10X Genomics single-cell RNA sequencing.

Single Cell RNA-Seq profiling of human embryonic kidney outer and inner cortical cells and kidney organoid cells

Lupus nephritis is a leading cause of mortality among systemic lupus erythematosus (SLE) patients, and its heterogeneous nature poses a significant challenge to the development of effective diagnostics and treatments. Single cell RNA sequencing (scRNA-seq) offers a potential solution to dissect the heterogeneity of the disease and enables the study of similar cell types distant from the site of renal injury to identify novel biomarkers. We applied scRNA-seq to human renal and skin biopsy tissues and demonstrated that scRNA-seq can be performed on samples obtained during routine care. Chronicity index, IgG deposition, and quantity of proteinuria correlated with a transcriptomic-based score composed of IFN-inducible genes in renal tubular cells. Furthermore, analysis of cumulative expression profiles of single cell keratinocytes dissociated from nonlesional, non–sun-exposed skin of patients with lupus nephritis also revealed upregulation of IFN-inducible genes compared with keratinocytes isolated from healthy controls. This indicates the possible use of scRNA-seq analysis of skin biopsies as a biomarker of renal disease. These data support the potential utility of scRNA-seq to provide new insights into the pathogenesis of lupus nephritis and pave the way for exploiting a readily accessible tissue to reflect injury in the kidney.

Single cell RNA sequencing to dissect the molecular heterogeneity in lupus nephritis

The integration of single cell transcriptome and chromatin accessibility datasets enables a deeper understanding of cell heterogeneity. We performed single nucleus ATAC (snATAC-seq) and RNA (snRNA-seq) sequencing to generate paired, cell-type-specific chromatin accessibility and transcriptional profiles of the adult human kidney. We demonstrate that snATAC-seq is comparable to snRNA-seq in the assignment of cell identity and can further refine our understanding of functional heterogeneity in the nephron. The majority of differentially accessible chromatin regions are localized to promoters and a significant proportion are closely associated with differentially expressed genes. Cell-type-specific enrichment of transcription factor binding motifs implicates the activation of NF-κB that promotes VCAM1 expression and drives transition between a subpopulation of proximal tubule epithelial cells. Our multi-omics approach improves the ability to detect unique cell states within the kidney and redefines cellular heterogeneity in the proximal tubule and thick ascending limb.

Single cell transcriptional and chromatin accessibility profiling redefine cellular heterogeneity in the adult human kidney

To define cellular mechanisms underlying kidney function and failure, the KPMP analyzes biopsy tissue in a multicenter research network to build cell-level process maps of the kidney. This study aimed to establish a single cell RNA sequencing strategy to use cell-level transcriptional profiles from kidney biopsies in KPMP to define molecular subtypes in glomerular diseases. Using multiple sources of adult human kidney reference tissue samples, 22,268 single cell profiles passed KPMP quality control parameters. Unbiased clustering resulted in 31 distinct cell clusters that were linked to kidney and immune cell types using specific cell markers. Focusing on endothelial cell phenotypes, in silico and in situ hybridization methods assigned 3 discrete endothelial cell clusters to distinct renal vascular beds. Transcripts defining glomerular endothelial cells (GEC) were evaluated in biopsies from patients with 10 different glomerular diseases in the NEPTUNE and European Renal cDNA Bank (ERCB) cohort studies. Highest GEC scores were observed in patients with focal segmental glomerulosclerosis (FSGS). Molecular endothelial signatures suggested 2 distinct FSGS patient subgroups with α-2 macroglobulin (A2M) as a key downstream mediator of the endothelial cell phenotype. Finally, glomerular A2M transcript levels associated with lower proteinuria remission rates, linking endothelial function with long-term outcome in FSGS.

Single cell transcriptomics identifies focal segmental glomerulosclerosis remission endothelial biomarker.

BackgroundRenal cell carcinoma (RCC) is the most common type of kidney cancer. Studying the pathogenesis of RCC is particularly important, because it could provide a direct guide for clinical treatment. Given that tumor heterogeneity is probably reflected at the mRNA level, the study of mRNA in RCC may reveal some potential tumor-specific markers, especially single-cell RNA sequencing (scRNA-seq).MethodsWe performed an exploratory study on three pathological types of RCC with a small sample size. This study presented clear-cell RCC (ccRCC), type 2 pRCC, and chRCC in a total of 30,263 high-quality single-cell transcriptome information from three pathological types of RCC. In addition, scRNA-seq was performed on normal kidneys. Tumor characteristics were well identified by the comparison between different pathological types of RCC and normal kidneys at the scRNA level.ResultsSome new tumor-specific markers for different pathologic types of RCC, such as SPOCK1, PTGIS, REG1A, CP and SPAG4 were identified and validated. We also discovered that NDUFA4L2 both highly expressed in tumor cells of ccRCC and type 2 pRCC. The presence of two different types of endothelial cells in ccRCC and type 2 pRCC was also identified and verified. An endothelial cell in ccRCC may be associated with fibroblasts and significantly expressed fibroblast markers, such as POSTN and COL3A1. At last, by applying scRNA-seq results, the activation of drug target pathways and sensitivity to drug responses was predicted in different pathological types of RCC.ConclusionsTaken together, these findings considerably enriched the single-cell transcriptomic information for RCC, thereby providing new insights into the diagnosis and treatment of RCC.

Single-Cell RNA Sequencing in Multiple Pathologic Types of Renal Cell Carcinoma Revealed Novel Potential Tumor-Specific Markers.

Urine passes through the entire kidney and urinary tract system starting from the glomerulus and ending to the urethra. Cells in the kidney and urinary tract could be exfoliated from the epithelium into the urine, while leukocyte could infiltrate from the local tissue into the urine, which makes the urine a useful subject for clinical evaluation of relevant diseases. We performed scRNA-seq on voided urine samples. 50–100 mL middle stream urine samples were collected from 12 Chinese healthy adults and combined for droplet-based single-cell RNA sequencing after flow cytometric sorting of live cells. We presented the first single-cell atlas of adult human urine and identified multiple previously unrecognized cell types. Based on our scRNA-seq analysis data, a SOX9+ cell population was identified in adult human urine which we speculated to have progenitor potential. Overall design: For scRNA-seq analysis, urine samples were collected from healthy adult volunteers. Midstream of voided urine specimen was collected for each donor. Samples were centrifuged immediately at the speed of 390 ×g and combined for droplet-based single-cell RNA sequencing after flow cytometric sorting of live cells.

Single-Cell RNA-Seq Analysis of cells from human urine

Lymphangioleiomyomatosis (LAM) is a metastasizing neoplasm of reproductive age women which causes cystic lung remodeling and progressive respiratory failure. While LAM lesions are known to contain abnormal smooth muscle-like cells which harbor mTOR activating mutations in TSC1 or TSC2, the tissue origins of the mutant “LAM cells” that invade the lung remain unclear. By employing single cell and single nuclear RNA sequencing on explanted LAM lungs, we identified a unique population of cells and associated signature genes and gene networks which were readily distinguished from those of endogenous lung cells. These unique LAMCORE cells shared closest transcriptomic similarity to normal uterus and share transcriptomic features with neural crest, as identified in uterine LAM lesions by single nuclear RNA-seq. Immunofluorescence microscopy demonstrated the expression of LAMCORE cell signature genes within LAM lesions in both lung and uterus. Serum aptamer proteomics and ELISA identified biomarkers consistent with the signature genes expressed and predicted to be secreted by LAMCORE cells. Single cell transcriptomics strongly supports a uterine neural crest origin of LAMCORE cells; providing insights into disease pathogenesis and informing future treatment strategies for LAM. Overall design: scRNA-seq and snRNA-seq was performed using the 10x Chromium platform on four lung explants from LAM patients undergoing lung transplantation. We also performed scRNA-seq on a renal AML resected from a patient with a sporadic AML (S-AML), and snRNA-seq on uterine tissue obtained at hysterectomy from an S-LAM patient. As controls, we conducted scRNA-seq on a 31 years old female explanted lung, brain dead, beating-heart, organ donor and snRNA-seq on one normal uterine tissue at hysterectomy from a 29 years old female patient with cervical cancer, and obtained scRNA-seq data of six additional female donor lungs from Gene Expression Omnibus (GSE122960) and scRNA-seq data from normal mouse uterus (GSE118180). qRT-PCR, immunofluorescence and immunochemical stains to validate the spatial relationships of LAM cells and LAMCORE signature genes.Serum aptamer proteomics and ELISA assays screening were used for validation of secretome predictions.

Single-Cell Transcriptomic Analysis Identifies a Unique Pulmonary Lymphangioleiomyomatosis Cell

IgA nephropathy (IgAN) is the leading cause of kidney failure due to an incomplete understanding of its pathogenesis. We perform single-cell RNA sequencing (RNA-seq) on kidneys and CD14+ peripheral blood mononuclear cells (PBMCs) collected from IgAN and normal samples. In IgAN, upregulation of JCHAIN in mesangial cells provides insight into the trigger mechanism for the dimerization and deposition of IgA1 in situ. The pathological mesangium also demonstrates a prominent inflammatory signature and increased cell-cell communication with other renal parenchymal cells and immune cells, suggesting disease progress from the mesangium to the entire kidney. Specific gene expression of kidney-resident macrophages and CD8+ T cells further indicates abnormal regulation associated with proliferation and inflammation. A transitional cell type among intercalated cells with fibrosis signatures is identified, suggesting an adverse outcome of interstitial fibrosis. Altogether, we systematically analyze the molecular events in the onset and progression of IgAN, providing a promising landscape for disease treatment.

Single-Cell Transcriptomics Reveal Immune Mechanisms of the Onset and Progression of IgA Nephropathy

Single cell genomics are revolutionizing our ability to characterize complex tissues. By contrast, the techniques used to analyze the renal biopsy are little changed over the past several decades. In this study we have tested the hypothesis that single cell RNA sequencing (sc-RNA-seq) can comprehensively describe cell types and states in a human kidney biopsy. We generated 8,746 single cell cell transcriptomes from a healthy adult kidney and a single kidney transplant biopsy core by sc-RNA-seq. Unsupervised clustering analysis of the biopsy identified 16 distinct cell types, including all of the major immune cell types and most native kidney cell types in this biopsy whose histologic read was mixed rejection. Endothelial cells formed three distinct sub-clusters: resting cells and two activated endothelial cell clusters, including expression of Fc-receptor pathway activation and immunoglobulin internalization genes, consistent with the pathologic diagnosis of antibody-mediated rejection. Monocytes formed two sub-clusters representing a non-classical CD16+ group and a classical CD16- group expressing dendritic cell maturation markers. The presence of both monocyte cell subtypes was validated by staining of of independent transplant biopsies. Comparison of healthy kidney epithelial transcriptomes to their biopsy counterparts identified novel segment specific pro-inflammatory responses. We mapped previously defined genes that associate with rejection outcomes to single cell types and generated a searchable online gene expression database. These findings represent a first step towards incorporation of single cell transcriptomics into kidney biopsy interpretation, describe a heterogeneous immune response in mixed rejection and provide a searchable resource for the scientific community.

Single-Cell Transcriptomics of a Human Kidney Allograft Biopsy Specimen Defines a Diverse Inflammatory Response

A comprehensive cellular anatomy of normal human kidney is vital to address the cellular origins of renal disease and renal cancer. Some kidney diseases may be cell type-specific, especially renal tubular cells. To investigate the classification and transcriptomic information of human kidney, we performed a method to obtain single-cell suspension of kidney rapidly, and conducted single-cell RNA sequencing (scRNA-seq). We present scRNA-seq data of 22,808 high quality cells from human kidneys of 3 donors. In this dataset, we show 13 clusters of normal human renal cells. Due to the high quality of single cell transcriptomic information, proximal tubule (PT) cells were classified into 6 subtypes and collecting ducts cells into 2 subtypes. Collectively, our data will provide a reliable reference for the studies of renal cell biology and kidney diseases.

Single-cell RNA sequencing of human kidney

Background: Acute kidney injury (AKI) is a common sequela of infection with SARS-CoV-2 and contributes to the severity and mortality from COVID-19. Here, we tested the hypothesis that kidney alterations induced by COVID-19-associated AKI could be detected in cells collected from urine. Methods: We performed single-cell RNA sequencing (scRNAseq) on cells recovered from the urine of eight hospitalized COVID-19 patients with (n=5) or without AKI (n=3) as well as four non-COVID-19 AKI patients (n=4) to assess differences in cellular composition and gene expression during AKI. Results: Analysis of 30,076 cells revealed a diverse array of cell types, most of which were kidney, urothelial, and immune cells. Pathway analysis of tubular cells from patients with AKI showed enrichment of transcripts associated with damage-related pathways compared to those without AKI. ACE2 and TMPRSS2 expression were highest in urothelial cells amongst cell types recovered. Notably, in one patient we detected SARS-CoV-2 viral RNA in urothelial cells. These same cells were enriched for transcripts associated with anti-viral and anti-inflammatory pathways. Conclusions: We successfully performed scRNAseq on urinary sediment from hospitalized patients with COVID-19 to noninvasively study cellular alterations associated with AKI and established a dataset that includes both injured and uninjured kidney cells. Additionally, we provide preliminary evidence of direct infection of urinary bladder cells by SARS-CoV-2. The urinary sediment contains a wealth of information and is a useful resource for studying the pathophysiology and cellular alterations that occur in kidney diseases. Overall design: Single-cell RNA sequencing of cells obtained from urine of patients with COVID-19. There are 12 total samples from 12 separate patients, 5 with COVID19 AKI, 3 with COVID19 without AKI, and 4 with non COVID19 AKI

Single-cell RNA sequencing of urinary cells reveals distinct cellular diversity in COVID-19-associated AKI

Diverse subtypes of renal cell carcinomas (RCCs) display a wide spectrum of histomorphologies, proteogenomic alterations, immune cell infiltration patterns, and clinical behavior. Delineating the cells of origin for different RCC subtypes will provide mechanistic insights into their diverse pathobiology. Here, we employed single-cell RNA sequencing (scRNA-seq) to develop benign and malignant renal cell atlases. Using a random forest model trained on this cell atlas, we predicted the putative cell of origin for more than 10 RCC subtypes. scRNA-seq also revealed several attributes of the tumor microenvironment in the most common subtype of kidney cancer, clear cell RCC (ccRCC). We elucidated an active role for tumor epithelia in promoting immune cell infiltration, potentially explaining why ccRCC responds to immune checkpoint inhibitors, despite having a low neoantigen burden. In addition, we characterized an association between high endothelial cell types and lack of response to immunotherapy in ccRCC. Taken together, these single-cell analyses of benign kidney and RCC provide insight into the putative cell of origin for RCC subtypes and highlight the important role of the tumor microenvironment in influencing ccRCC biology and response to therapy.

Single-cell analyses of renal cell cancers reveal insights into tumor microenvironment, cell of origin, and therapy response

Knowledge of the transcriptional programs underpinning the functions of human kidney cell populations at homeostasis is limited. We present a single-cell perspective of healthy human kidney from 19 living donors, with equal contribution from males and females, profiling the transcriptome of 27677 cells to map human kidney at high resolution. Sex-based differences in gene expression within proximal tubular cells were observed, specifically, increased anti-oxidant metallothionein genes in females and aerobic metabolism-related genes in males. Functional differences in metabolism were confirmed in proximal tubular cells, with male cells exhibiting higher oxidative phosphorylation and higher levels of energy precursor metabolites. We identified kidney-specific lymphocyte populations with unique transcriptional profiles indicative of kidney-adapted functions. Significant heterogeneity in myeloid cells was observed, with a MRC1+LYVE1+FOLR2+C1QC+ population representing a predominant population in healthy kidney. This study provides a detailed cellular map of healthy human kidney, and explores the complexity of parenchymal and kidney-resident immune cells.

Single-cell profiling of healthy human kidney reveals features of sex-based transcriptional programs and tissue-specific immunity

Clear cell renal carcinoma (ccRCC) is a heterogeneous disease with a variable post-surgical course. To assemble a comprehensive ccRCC tumor microenvironment (TME) atlas, we performed single-cell RNA sequencing (scRNA-seq) of hematopoietic and non-hematopoietic subpopulations from tumor and tumor-adjacent tissue of treatment-naive ccRCC resections. We leveraged the VIPER algorithm to quantitate single-cell protein activity and validated this approach by comparison to flow cytometry. The analysis identified key TME subpopulations, as well as their master regulators and candidate cell-cell interactions, revealing clinically relevant populations, undetectable by gene-expression analysis. Specifically, we uncovered a tumor-specific macrophage subpopulation characterized by upregulation of TREM2/APOE/C1Q, validated by spatially resolved, quantitative multispectral immunofluorescence. In a large clinical validation cohort, these markers were significantly enriched in tumors from patients who recurred following surgery. The study thus identifies TREM2/APOE/C1Q-positive macrophage infiltration as a potential prognostic biomarker for ccRCC recurrence, as well as a candidate therapeutic target.

Single-cell protein activity analysis identifies recurrence-associated renal tumor macrophages.

Clear cell renal cell carcinomas (ccRCCs) are highly immune infiltrated, but the effect of immune heterogeneity on clinical outcome in ccRCC has not been fully characterized. Here we perform paired single-cell RNA (scRNA) and T cell receptor (TCR) sequencing of 167,283 cells from multiple tumor regions, lymph node, normal kidney, and peripheral blood of two immune checkpoint blockade (ICB)-naïve and four ICB-treated patients to map the ccRCC immune landscape. We detect extensive heterogeneity within and between patients, with enrichment of CD8A+ tissue-resident T cells in a patient responsive to ICB and tumor-associated macrophages (TAMs) in a resistant patient. A TCR trajectory framework suggests distinct T cell differentiation pathways between patients responding and resistant to ICB. Finally, scRNA-derived signatures of tissue-resident T cells and TAMs are associated with response to ICB and targeted therapies across multiple independent cohorts. Our study establishes a multimodal interrogation of the cellular programs underlying therapeutic efficacy in ccRCC.

Single-cell sequencing links multiregional immune landscapes and tissue-resident T cells in ccRCC to tumor topology and therapy efficacy.

Messenger RNA encodes cellular function and phenotype. In the context of human cancer, it defines the identities of malignant cells and the diversity of tumor tissue. We studied 72,501 single-cell transcriptomes of human renal tumors and normal tissue from fetal, pediatric, and adult kidneys. We matched childhood Wilms tumor with specific fetal cell types, thus providing evidence for the hypothesis that Wilms tumor cells are aberrant fetal cells. In adult renal cell carcinoma, we identified a canonical cancer transcriptome that matched a little-known subtype of proximal convoluted tubular cell. Analyses of the tumor composition defined cancer-associated normal cells and delineated a complex vascular endothelial growth factor (VEGF) signaling circuit. Our findings reveal the precise cellular identities and compositions of human kidney tumors.

Single-cell transcriptomes from human kidneys reveal the cellular identity of renal tumors.

Kidneys have one of the most complex three-dimensional cellular organizations in the body, but the spatial molecular principles of kidney health and disease are poorly understood. Here we generate high-quality single cell (sc), single nuclear (sn), spatial (sp) RNA expression and sn open chromatin datasets for 73 samples, capturing half a million cells from healthy, diabetic, and hypertensive diseased human kidneys. Combining the sn/sc and sp RNA information, we identify > 100 cell types and states and successfully map them back to their spatial locations. Computational deconvolution of spRNA-seq identifies glomerular/vascular, tubular, immune, and fibrotic spatial microenvironments (FMEs). Although injured proximal tubule cells appear to be the nidus of fibrosis, we reveal the complex, heterogenous cellular and spatial organization of human FMEs, including the highly intricate and organized immune environment. We demonstrate the clinical utility of the FME spatial gene signature for the classification of a large number of human kidneys for disease severity and prognosis. We provide a comprehensive spatially-resolved molecular roadmap for the human kidney and the fibrotic process and demonstrate the clinical utility of spatial transcriptomics.

Spatially resolved human kidney multi-omics single cell atlas highlights the key role of the fibrotic microenvironment in kidney disease progression

Tissue-resident immune cells are important for organ homeostasis and defense. The epithelium may contribute to these functions directly, or via crosstalk with immune cells. We resolved the spatio-temporal immune topology of the human kidney with a scRNAseq dataset from 40,268 mature and 27,203 fetal kidney cells. Within the epithelial compartment there were zonated patterns of immune gene expression, with pelvic epithelial anti-microbial peptide expression in mature but not fetal kidneys. Tissue-resident immune cells were evident in both fetal and mature kidney, with post-natal acquisition of infection-defense capabilities. Epithelial-immune signalling orchestrated localization of macrophages and neutrophils to regions of the kidney susceptible to infection.

Spatio-temporal immune zonation of the human kidney

We have created a compendium of single cell transcriptome data from the model
organism Mus musculus comprising more than 100,000 cells from 20 organs and
tissues. These data represent a new resource for cell biology, revealing gene
expression in poorly characterized cell populations and allowing for direct and
controlled comparison of gene expression in cell types shared between tissues, such
as T-lymphocytes and endothelial cells from distinct anatomical locations. Two
distinct technical approaches were used for most tissues: one approach, microfluidic
droplet-based 3’-end counting, enabled the survey of thousands of cells at relatively
low coverage, while the other, FACS-based full length transcript analysis, enabled
characterization of cell types with high sensitivity and coverage. The cumulative
data provide the foundation for an atlas of transcriptomic cell biology.

Tabula Muris: Transcriptomic characterization of 20 organs and tissues from Mus musculus at single cell resolution

In recent years there has been tremendous progress towards deep molecular characterization of cell types using single cell transcriptome sequencing. Here we report a single cell transcriptomic atlas comprising nearly 500,000 cells from 24 different human tissues and organs. In several instances multiple organs were analyzed from the same donor. Analyzing organs from the same individual controls for genetic background, age, environment, and epigenetic effects, and enables a detailed comparison of cell types that are shared between tissues. This resource provides a rich molecular characterization of more than 400 cell types, their distribution across tissues, and detailed information about tissue specific variation in gene expression. We have used the fact that multiple tissues came from the same donor to study the clonal distribution of T cells between tissues, to understand the tissue specific mutation rate in B cells, and to analyze the cell cycle state and proliferative potential of shared cell types across tissues. Finally, we have also used this data to characterize cell type specific RNA splicing and how such splicing varies across tissues within an individual.

The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

The immune cell landscape in kidneys of patients with lupus nephritis.

Diabetic nephropathy is characterized by damage to both the glomerulus and tubulointerstitium, but relatively little is known about accompanying cell-specific changes in gene expression. We performed unbiased single nucleus RNA sequencing (snRNAseq) on cryopreserved human diabetic kidney samples to generate 23,980 single nucleus transcriptomes from three control and three early diabetic nephropathy samples. All major cell types of the kidney were represented in the final dataset. Side by side comparison demonstrated cell-type-specific changes in gene expression that are important for ion transport, angiogenesis, and immune cell activation. In particular, we show that the diabetic thick ascending limb, late distal convoluted tubule, and principal cells all adopt a gene expression signature consistent with increased potassium secretion, including alterations in Na-K+-ATPase, WNK1, mineralocorticoid receptor and NEDD4L expression, as well as decreased paracellular calcium and magnesium reabsorption. We also identify strong angiogenic signatures in glomerular cell types, proximal convoluted tubule, distal convoluted tubule and principal cells. Taken together, these results suggest that increased potassium secretion and angiogenic signaling represent early kidney responses in human diabetic nephropathy.

The single-cell transcriptomic landscape of early human diabetic nephropathy

Tissue engineering offers a promising treatment strategy for ureteral strictures. Successful ureter engineering is necessitated by detailed understanding of the tissue architecture, cellular heterogeneity, and signaling pathways underlying regeneration. We define and spatially map cell populations within the human ureter by using a combinatorial approach: single-cell RNA sequencing, 10X Visium spatial transciptomics, and immunofluorescence. The stromal and urothelial cell populations are analyzed in detail, and we infer potential cell-cell communication networks underpinning the bi-directional crosstalk between these compartments. Specifically, we analyze and experimentally validate the importance of Sonic Hedgehog (SHH) signaling pathway in adult stem cell maintenance. The SHH-expressing basal cells support organoid generation in vitro, and accurately predict the differentiation trajectory of basal stem cells, to terminally differentiated umbrella cells, in vivo. Our results highlight essential processes involved in adult ureter tissue homeostasis, and provide a toolkit for guiding ureter tissue engineering. Overall design: Two normal human ureters each were subjected to spatial transcriptomics using 10X Genomics Spatial Gene Expression. Data was processed using Spaceranger 1.3.0 and anayzed using Seurat v4.0.4 to better understand the architecture and cellular heterogeneity of the ureter. These samples have matching single cell RNAseq data, which were used to enhance and validate the Spatial Gene Expression data.

Kidney Network

Network Description

Kidney Network atlases are still under development.

Network Coordinators