Targeted Single-Cell RNA-seq Identifies Minority Cell Types of Kidney Distal Nephron.
04 Mar 2021-Journal of The American Society of Nephrology (J Am Soc Nephrol)-Vol. 32, Iss: 4, pp 886-896
TL;DR: In this paper, single-cell transcriptomics revealed unexpected diversity among the cells of the distal nephron in mouse, with orthogonally variable expression of Slc8a1, Calb1, and Ckb.
Abstract: Background Proximal tubule cells dominate the kidney parenchyma numerically, although less abundant cell types of the distal nephron have disproportionate roles in water and electrolyte balance. Methods Coupling of a FACS-based enrichment protocol with single-cell RNA-seq profiled the transcriptomes of 9099 cells from the thick ascending limb (CTAL)/distal convoluted tubule (DCT) region of the mouse nephron. Results Unsupervised clustering revealed Slc12a3 +/Pvalb + and Slc12a3 +/Pvalb - cells, identified as DCT1 and DCT2 cells, respectively. DCT1 cells appear to be heterogeneous, with orthogonally variable expression of Slc8a1, Calb1, and Ckb. An additional DCT1 subcluster showed marked enrichment of cell cycle-/cell proliferation-associated mRNAs (e.g., Mki67, Stmn1, and Top2a), which fit with the known plasticity of DCT cells. No DCT2-specific transcripts were found. DCT2 cells contrast with DCT1 cells by expression of epithelial sodium channel β- and γ-subunits and much stronger expression of transcripts associated with calcium transport (Trpv5, Calb1, S100g, and Slc8a1). Additionally, scRNA-seq identified three distinct CTAL (Slc12a1 +) cell subtypes. One of these expressed Nos1 and Avpr1a, consistent with macula densa cells. The other two CTAL clusters were distinguished by Cldn10 and Ptger3 in one and Cldn16 and Foxq1 in the other. These two CTAL cell types were also distinguished by expression of alternative Iroquois homeobox transcription factors, with Irx1 and Irx2 in the Cldn10 + CTAL cells and Irx3 in the Cldn16 + CTAL cells. Conclusions Single-cell transcriptomics revealed unexpected diversity among the cells of the distal nephron in mouse. Web-based data resources are provided for the single-cell data.
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TL;DR: In this article, a full-length, small-sample RNA-seq protocol profiled transcriptomes for all 14 renal tubule segments microdissected from mouse kidneys and identified >34,000 transcripts, including 3709 that were expressed in a segment-specific manner.
Abstract: Background The repertoire of protein expression along the renal tubule depends both on regulation of transcription and regulation of alternative splicing that can generate multiple proteins from a single gene. Methods A full-length, small-sample RNA-seq protocol profiled transcriptomes for all 14 renal tubule segments microdissected from mouse kidneys. Results This study identified >34,000 transcripts, including 3709 that were expressed in a segment-specific manner. All data are provided as an online resource (https://esbl.nhlbi.nih.gov/MRECA/Nephron/). Many of the genes expressed in unique patterns along the renal tubule were solute carriers, transcription factors, or G protein-coupled receptors that account for segment-specific function. Mapping the distribution of transcripts associated with Wnk-SPAK-PKA signaling, renin-angiotensin-aldosterone signaling, and cystic diseases of the kidney illustrated the applications of the online resource. The method allowed full-length mapping of RNA-seq reads, which facilitated comprehensive, unbiased characterization of alternative exon usage along the renal tubule, including known isoforms of Cldn10, Kcnj1 (ROMK), Slc12a1 (NKCC2), Wnk1, Stk39 (SPAK), and Slc14a2 (UT-A urea transporter). It also identified many novel isoforms with segment-specific distribution. These included variants associated with altered protein structure (Slc9a8, Khk, Tsc22d1, and Scoc), and variants that may affect untranslated, regulatory regions of transcripts (Pth1r, Pkar1a, and Dab2). Conclusions Full-length, unbiased sequencing of transcripts identified gene-expression patterns along the mouse renal tubule. The data, provided as an online resource, include both quantitative and qualitative differences in transcripts. Identification of alternative splicing along the renal tubule may prove critical to understanding renal physiology and pathophysiology.
98 citations
University of California, Berkeley1, University of Michigan2, University of Nebraska Medical Center3, Harvard University4, Indiana University5, Washington University in St. Louis6, Broad Institute7, Boston University8, Yale University9, University of Pittsburgh10, Columbia University11, Cleveland Clinic12, University of Texas Southwestern Medical Center13, Johns Hopkins University14
TL;DR: In this paper, a high-resolution cellular atlas of 100 cell types and states, their associated molecular profiles, and interactions within tissue neighborhoods was provided. But, the authors did not identify and define cellular states altered in kidney injury, encompassing cycling, adaptive or maladaptive repair, transitioning and degenerative states affecting several segments.
Abstract: Understanding kidney disease relies upon defining the complexity of cell types and states, their associated molecular profiles, and interactions within tissue neighborhoods. We have applied multiple single-cell or -nucleus assays (>400,000 nuclei/cells) and spatial imaging technologies to a broad spectrum of healthy reference (n = 42) and disease (n = 42) kidneys. This has provided a high resolution cellular atlas of 100 cell types that include rare and novel cell populations. The multi-omic approach provides detailed transcriptomic profiles, epigenomic regulatory factors, and spatial localizations for major cell types spanning the entire kidney. We further identify and define cellular states altered in kidney injury, encompassing cycling, adaptive or maladaptive repair, transitioning and degenerative states affecting several segments. Molecular signatures of these states permitted their localization within injury neighborhoods using spatial transcriptomics, and large-scale 3D imaging analysis of ∼1.2 million neighborhoods provided linkages to active immune responses. These analyses further defined biological pathways relevant to injury niches, including signatures underlying the transition from reference to predicted maladaptive states that were associated with a decline in kidney function during chronic kidney disease. This human kidney cell atlas, including injury cell states and neighborhoods, will be a valuable resource for future studies.
40 citations
TL;DR: For example, this article found that there are at least 16 different highly specialized epithelial cell types in the mammalian kidney and the number of specialized endothelial cells, immune cells, and interstitial cell types might even be larger.
Abstract: The kidney maintains electrolyte, water, and acid-base balance, eliminates foreign and waste compounds, regulates blood pressure, and secretes hormones. There are at least 16 different highly specialized epithelial cell types in the mammalian kidney. The number of specialized endothelial cells, immune cells, and interstitial cell types might even be larger. The concerted interplay between different cell types is critical for kidney function. Traditionally, cells were defined by their function or microscopical morphological appearance. With the advent of new single-cell modalities such as transcriptomics, epigenetics, metabolomics, and proteomics we are entering into a new era of cell type definition. This new technological revolution provides new opportunities to classify cells in the kidney and understand their functions.
31 citations
TL;DR: In this article , an integrated reference map of cells, pathways, and genes using unaffected regions of nephrectomy tissues and undiseased human biopsies from 56 adult subjects is presented.
Abstract: Kidney Precision Medicine Project (KPMP) is building a spatially specified human kidney tissue atlas in health and disease with single-cell resolution. Here, we describe the construction of an integrated reference map of cells, pathways, and genes using unaffected regions of nephrectomy tissues and undiseased human biopsies from 56 adult subjects. We use single-cell/nucleus transcriptomics, subsegmental laser microdissection transcriptomics and proteomics, near-single-cell proteomics, 3D and CODEX imaging, and spatial metabolomics to hierarchically identify genes, pathways, and cells. Integrated data from these different technologies coherently identify cell types/subtypes within different nephron segments and the interstitium. These profiles describe cell-level functional organization of the kidney following its physiological functions and link cell subtypes to genes, proteins, metabolites, and pathways. They further show that messenger RNA levels along the nephron are congruent with the subsegmental physiological activity. This reference atlas provides a framework for the classification of kidney disease when multiple molecular mechanisms underlie convergent clinical phenotypes.
29 citations
TL;DR: Slide-seqV2 as mentioned in this paper was applied to mouse and human kidneys, in healthy and distinct disease paradigms, and it revealed a cell neighborhood centered around a population of LYVE1+ macrophages.
22 citations
References
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TL;DR: An overview of DAMP-sensing receptors is provided, the crosstalk between these receptors is discussed, and the cross-regulation of these receptors and their ligands are discussed.
Abstract: The innate immune system has the capacity to detect ‘non-self’ molecules derived from pathogens, known as pathogen-associated molecular patterns, via pattern recognition receptors. In addition, an increasing number of endogenous host-derived molecules, termed damage-associated molecular patterns (DAMPs), have been found to be sensed by various innate immune receptors. The recognition of DAMPs, which are produced or released by damaged and dying cells, promotes sterile inflammation, which is important for tissue repair and regeneration, but can also lead to the development of numerous inflammatory diseases, such as metabolic disorders, neurodegenerative diseases, autoimmune diseases and cancer. Here we examine recent discoveries concerning the roles of DAMP-sensing receptors in sterile inflammation and in diseases resulting from dysregulated sterile inflammation, and then discuss insights into the cross-regulation of these receptors and their ligands. Host-derived molecules, the so-called damage-associated molecular patterns (DAMPs), can induce sterile inflammation. This Review provides an overview of DAMP-sensing receptors, discusses the crosstalk between these receptors and explores their role in disease.
762 citations
TL;DR: It is inferred that inherited kidney diseases that arise from distinct genetic mutations but share the same phenotypic manifestation originate from the same differentiated cell type, and that the collecting duct in kidneys of adult mice generates a spectrum of cell types through a newly identified transitional cell.
Abstract: Our understanding of kidney disease pathogenesis is limited by an incomplete molecular characterization of the cell types responsible for the organ’s multiple homeostatic functions. To help fill this knowledge gap, we characterized 57,979 cells from healthy mouse kidneys by using unbiased single-cell RNA sequencing. On the basis of gene expression patterns, we infer that inherited kidney diseases that arise from distinct genetic mutations but share the same phenotypic manifestation originate from the same differentiated cell type. We also found that the collecting duct in kidneys of adult mice generates a spectrum of cell types through a newly identified transitional cell. Computational cell trajectory analysis and in vivo lineage tracing revealed that intercalated cells and principal cells undergo transitions mediated by the Notch signaling pathway. In mouse and human kidney disease, these transitions were shifted toward a principal cell fate and were associated with metabolic acidosis.
751 citations
TL;DR: The highly conserved Notch signalling pathway functions in many different developmental and homeostatic processes, which raises the question of how this pathway can achieve such diverse outcomes.
Abstract: The highly conserved Notch signalling pathway functions in many different developmental and homeostatic processes, which raises the question of how this pathway can achieve such diverse outcomes. With a direct route from the membrane to the nucleus, the Notch pathway has fewer opportunities for regulation than do many other signalling pathways, yet it generates exquisitely patterned structures, including sensory hair cells and branched arterial networks. More confusingly, its activity promotes tissue growth and cancers in some circumstances but cell death and tumour suppression in others. Many different regulatory mechanisms help to shape the activity of the Notch pathway, generating functional outputs that are appropriate for each context. These mechanisms include the receptor-ligand landscape, the tissue topology, the nuclear environment and the connectivity of the regulatory networks.
725 citations
TL;DR: The two major components of the kidney, the collecting system and the nephron, have different developmental histories, and their embryological origin and the genes controlling their morphogenesis, patterning, and differentiation are considered.
624 citations
TL;DR: It is concluded that nitric oxide synthase in macula densa cells is activated by tubular-fluid reabsorption and mediates a vasodilating component to the tubuloglomerular feedback response.
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527 citations