Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

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.

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Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease

Although we can increasingly measure transcription, chromatin, methylation, and other aspects of molecular biology at single-cell resolution, most assays survey only one aspect of cellular biology. Here we describe sci-CAR, a combinatorial indexing–based coassay that jointly profiles chromatin accessibility and mRNA (CAR) in each of thousands of single cells. As a proof of concept, we apply sci-CAR to 4825 cells, including a time series of dexamethasone treatment, as well as to 11,296 cells from the adult mouse kidney. With the resulting data, we compare the pseudotemporal dynamics of chromatin accessibility and gene expression, reconstruct the chromatin accessibility profiles of cell types defined by RNA profiles, and link cis-regulatory sites to their target genes on the basis of the covariance of chromatin accessibility and transcription across large numbers of single cells.

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

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

Tumours comprise a heterogeneous collection of cells with distinct genetic and phenotypic properties that can differentially promote progression, metastasis and drug resistance. Emerging single-cell technologies provide a new opportunity to profile individual cells within tumours and investigate what roles they play in these processes. This Review discusses key technological considerations for single-cell studies in cancer, new findings using single-cell technologies and critical open questions for future applications.

Tumour heterogeneity and metastasis at single-cell resolution.

Prior RNA sequencing (RNA-seq) studies have identified complete transcriptomes for most renal epithelial cell types. The exceptions are the cell types that make up the renal collecting duct, namely intercalated cells (ICs) and principal cells (PCs), which account for only a small fraction of the kidney mass, but play critical physiological roles in the regulation of blood pressure, extracellular fluid volume, and extracellular fluid composition. To enrich these cell types, we used FACS that employed well-established lectin cell surface markers for PCs and type B ICs, as well as a newly identified cell surface marker for type A ICs, c-Kit. Single-cell RNA-seq using the IC- and PC-enriched populations as input enabled identification of complete transcriptomes of A-ICs, B-ICs, and PCs. The data were used to create a freely accessible online gene-expression database for collecting duct cells. This database allowed identification of genes that are selectively expressed in each cell type, including cell-surface receptors, transcription factors, transporters, and secreted proteins. The analysis also identified a small fraction of hybrid cells expressing aquaporin-2 and anion exchanger 1 or pendrin transcripts. In many cases, mRNAs for receptors and their ligands were identified in different cells (e.g., Notch2 chiefly in PCs vs. Jag1 chiefly in ICs), suggesting signaling cross-talk among the three cell types. The identified patterns of gene expression among the three types of collecting duct cells provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.

Transcriptomes of major renal collecting duct cell types in mouse identified by single-cell RNA-seq.

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.

A Comprehensive Map of mRNAs and Their Isoforms across All 14 Renal Tubule Segments of Mouse.

https://www.kidney-international.org/article/S0085-2538(18)30912-8/pdf

Representation and relative abundance of cell-type selective markers in whole-kidney RNA-Seq data.

The renal collecting duct in the mammalian kidney plays a critical role in the regulation of extracellular volume, osmolarity, and pH. It contains at least three structurally and functionally distinct cell types: principal cells (PCs), α-intercalated cells (α-ICs), and β- intercalated cells (β-ICs).1 PCs regulate Na+ and water homeostasis. α-ICs and β-ICs are involved in acid and bicarbonate secretion, respectively.2,3 Aqp2 is a well accepted PC marker, whereas carbonic anhydrase type II (CAII) is expressed in all ICs.4,5 The proton-pumping V-ATPase is a complex, multisubunit enzyme consisting of at least 13 subunits, including A, B1, and B2. It is highly expressed in the plasma membranes of some epithelial cells, including ICs in the kidney.6 The kidney variant of the band 3 Cl−/HCO3− exchanger (AE1) and the sodium-independent chloride/iodide transporter Pendrin are present in α-IC and β-IC, respectively.7

β-ICs in primary culture behave as stem cells and give rise to α-ICs, PCs, and PC/IC “hybrids,” based on functional characteristics and expression of IC and PC markers. Unlike β-IC, sorted PCs maintain their PC phenotype as terminally differentiated cells.8 Metabolic acidosis reduced β-ICs and increased α-ICs without changing the total IC population, indicating β-IC conversion to α-IC.9 Mice lacking the extracellular matrix protein hensin/DMBT1 in ICs contained no typical α-ICs.10 Inactivation of the forkhead transcription factor Foxi1 resulted in a single cell type (Aqp2+ CAII+) that lacks other IC markers (Pendrin, AE1, AE4, and V-ATPase B1) in the cortex and medulla, raising the possibility that PCs and ICs are differentiated from epithelial precursor cells characterized by Foxi1− Aqp2+ CAII+. 11 Both hesin/DMBT1 and Foxi1 mutant mice develop distal renal tubular acidosis. In CAII-deficient mice, ICs (evaluated by AE1 and H+-ATPase expression) were reduced and replaced by PCs.4 However, whether ICs are derived from PCs or Aqp2+ progenitor cells has not been demonstrated.

Disruptor of telomeric silencing (Dot1) was first cloned in yeast as a gene affecting telomeric silencing.12 Dot1 and its mammalian homologues (Dot1l) encode a methyltransferase specific for histone H3 K79.13–15 Dot1l regulates transcription, development, erythropoiesis, differentiation, proliferation, and leukemogenesis.16–18 Global deletion of Dot1l causes embryonic lethality.19

We previously cloned mouse Dot1l, observed high mRNA expression in mouse kidney and strong H3 dimethyl K79 (m2K79) staining in PC and IC, and identified five alternative splicing variants (Dot1a-e).15,20 Our work also revealed that Dot1a regulates mRNA expression and activity of the epithelial Na+ channel in mouse cortical collecting duct M1 and mouse inner medulla collecting duct line 3 (IMCD3) cells.20–22 However, the function of Dot1l in vivo in mouse kidney has not been directly addressed.

In this study, we used our Aqp2:Cre and newly developed Dot1lf/f mice to generate connecting tube/collecting duct (CNT/CD)–specific Dot1l-deficient (Dot1lf/f Aqp2:Cre or Dot1lAC) mice and then determined whether Dot1l deletion affects PC/IC differentiation. To our knowledge, this study provides for the first time direct in vivo evidence that ICs as defined by standard markers can be derived from Aqp2-expressing cells. Derived ICs are observed in both developing and adult kidneys. Dot1l deletion facilitates this process, leading to an increase in IC/PC ratio and urine volume. This study highlights the plasticity of renal tubular epithelia, the origin of most ICs from Aqp2+ cells, and the significance of Dot1l inactivation in renal physiology and pathology.

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Aqp2-Expressing Cells Give Rise to Renal Intercalated Cells

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Lihe Chen

Papers

Transcriptomes of major renal collecting duct cell types in mouse identified by single-cell RNA-seq.

A Comprehensive Map of mRNAs and Their Isoforms across All 14 Renal Tubule Segments of Mouse.

Representation and relative abundance of cell-type selective markers in whole-kidney RNA-Seq data.

Aqp2-Expressing Cells Give Rise to Renal Intercalated Cells

Renal-Tubule Epithelial Cell Nomenclature for Single-Cell RNA-Sequencing Studies.