REVIEW www.jasn.org Renal-Tubule Epithelial Cell Nomenclature for Single-Cell RNA-Sequencing Studies Lihe Chen,1 Jevin Z. Clark,1 Jonathan W. Nelson,2 Brigitte Kaissling,3 David H. Ellison ,2 and Mark A. Knepper1 1Epithelial Systems Biology Laboratory, Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; 2Division of Nephrology and Hypertension, Oregon Health & Science University, Portland, Oregon; and 3Institute of Anatomy, University of Zurich, Zurich, Switzerland The assignment of cell type names to also include information on the per- the International Union of Physiologic individual cell clusters in single-cell centage of each epithelial cell type in Sciences and published in in Kidney RNA-sequencing (RNA-seq; including mouse kidney and on widely accepted International in 1988.3 The 1988 nomen- single-nucleus RNA-seq) studies is a mRNA/protein markers that are consid- clature was focused on what to call each critical bioinformatic step because re- ered prerequisites for identification of renal tubule segment, and not neces- searchers will need to be able to reliably individual cell types. sarily what to call individual cell types. map the pertinent transcriptomes to The mammalian renal tubule is made Accordingly, we refocused the recom- other types of information in the litera- up of at least 14 segments, containing at mended terminology to the individual ture. Ambiguous or redundant cell-type least 16 distinct epithelial cell types.4–6 cell types present in the various tubule names would impede such mapping Each cell type has its own characteristic segments. tasks. The solution to this problem is to set of cellular functions (mostly related Before presenting the list of recom- create “controlled vocabularies”1,2 that to transport and metabolism), which mended cell-type terminology, we sum- can be preferentially used in reports of have been elucidated largely over the marize, in a greatly simplified manner, single-cell RNA-seq data and other types past 50 years since the development of the current procedures for mapping of -omic data at a single-cell level. In single-tubule microdissection approaches gene expression lists from single-cell this review, we provide such a controlled by Burg et al.7 and the expansion of the RNA-seq to the various known cell types. vocabulary for the epithelial cells that micropuncture technique to mamma- A single-cell RNA-seq dataset contains make up the mammalian renal tubule. lian physiology.8,9 Furthermore, each (for each cell) a list of expressed genes The nomenclature list was not produced cell type has its own unique transcrip- together with some measure of their de novo in this review, but instead repre- tome as elucidated by recent RNA-seq mRNA expression levels. The initial sents an adaptation of a prior, widely ac- studies carried out at the level of single task, carried out independently of cell cepted nomenclature list for renal tubule microdissected tubules6 and single type assignment, is to cluster the cells segments published in 1988 in Kidney cells.10–26 The new transcriptomic data into groups that are similar on the basis International.3 This was done because promises to expand and sharpen our un- of the lists of their expressed mRNAs historical consistency is important to as- derstanding of renal tubule function, as along with their expression levels. These “ ” sure that names assigned to clusters in the data are mapped to cellular processes clusters are then mapped to cell-type single-cell RNA-seq studies mesh well responsible for the macroscopic function names that are assigned on the basis of with the “pregenomic” 20th century lit- of each renal tubule segment. erature, which provides robust and well To allow this mapping to be done ac- validated information on kidney cell curately, it is imperative that the cell-type Published online ahead of print. Publication date structure and function. The 1988 article terminology used for single-tubule and available at www.jasn.org. offered several alternative names for single-cell gene expression studies be Correspondence: Dr. Mark A. Knepper, Epithelial some of the segments and cell types. consistent with terminology used in the Systems Biology Laboratory, Systems Biology Our strategy here is to propose nomen- functional and biochemical studies done Center, National Heart, Lung, and Blood Institute, fl National Institutes of Health, MSC-1603, 10 Center claturethatre ects the most common in the past. Here, we summarize recom- Drive, Bethesda, MD 20892-1603. Email: knep@ usage from 1988 to the present in each mended terminology for renal tubule helix.nih.gov case. In this report, we not only describe epithelial cells derived from prior recom- Copyright © 2019 by the American Society of preferred names for each cell type, but mendations by the Renal Commission of Nephrology JASN 30: ccc–ccc,2019 ISSN : 1046-6673/3008-ccc 1 REVIEW www.jasn.org representation of cell-type selective Ontology (RECON) webpage (https:// the latter two junctions are easily recog- marker genes. The problem we are ad- hpcwebapps.cit.nih.gov/ESBL/Database/ nizable macroscopically, but the corti- dressing in this review is that there is RECOn/). Figure 1 shows both a short-loop comedullary junction is obscure, at least presently no generally accepted “con- and a long-loop nephron superimposed on in rodents, without benefitofastereo- trolled vocabulary” for renal tubule epi- regional boundaries of the kidney. The struc- microscope. Here, we present the rationale thelial cell types, i.e., there is no standard tures that define the regional boundaries are for the terms presented, some alternative list of acceptable terms. Consequently, the arcuate arteries (corticomedullary junc- terminology, and some alternative terms to terminology has been inconsistent among tion), the transition from proximal straight be avoided. individual single-cell RNA-seq articles,10–26 tubules (PSTs) to descending thin limbs and the correct mappings to functional and (DTLs) of the loops of Henle (inner/outer biochemical studies done in the pregenomic stripe of outer medulla junction) and the PROXIMAL TUBULE era are uncertain. transition from the ascending thin limbs NOMENCLATURE Recommended terms for renal tubule of Henle of the long-loop nephrons to epithelial cell types in kidneys of mature the medullary thick ascending limbs There are two sets of terms commonly mice, rats, or humans are given in Figure 1 (MTALs) of the loops of Henle (inner- used to describe proximal tubule cells. and are listed on the Renal Epithelial Cell outer medullary junction).4 Generally, One divides the proximal tubule into three Figure 1. Renal tubule cell nomenclature. The scheme shows the connection of both a short-looped nephron and a long-looped nephron to the collecting duct system. The numbers point to different renal tubule segments and the cell types that they contain are listed on the rightof thescheme.Dashedlines indicateregional boundaries.The corticomedullary junction isdefinedbythearcuate arteries(not showninthisdiagram).Thedivisionbetweentheouter stripe and inner stripe of the outer medulla is defined by the transitions from PSTs to DTLs of the loops of Henle. The junction between the outer medulla and inner medulla is defined by the transitions from the ascending thin limbs (ATL) of Henle of the long-loop nephrons to the MTALs of the loops of Henle. The division between the cortical labyrinth and cortical medullary rays is indicated but not labeled. Drawing is on the basis of the original Renal Commission of the In- ternational Union of Physiologic Sciences publication.3 2 JASN JASN 30: ccc–ccc,2019 www.jasn.org REVIEW segments called S1, S2, and S3 on the basis topologically distinct from the other three considered to include the aquaporin-2 of differences in morphology, topology, thin limb segments.29 All of the thin limb (AQP2) expressing cells of the collecting and location in the kidney.27,28 Function- segments are difficult to microdissect from duct. Hence, the term distal tubule is am- ally, the S2 is recognized as the segment the kidney and difficult to study by mi- biguous and should be avoided in RNA-seq responsible for para-aminohippurate se- cropuncture, limiting the amount of studies. Another similar term is “distal cretion and, by implication, responsible functional information we have about nephron,” commonly used to designate for general organic anion and cation se- them. the portion of the renal tubule from the cretion.28 A limitation of this classifica- In contrast, the thick ascending limb macula densa through the end of the inner tion is the lack of a discrete transition (TAL) is relatively easy to study in iso- medullary collecting duct (IMCD), thereby from one to the other morphologically. lation and has well defined functional comprised of multiple cell types. This lack of clear transitions suggests roles.30–32 The TAL is normally divided Rabbits exhibit a distinct transition that there are probably intermediate cell into the MTAL and the CTAL on the point at the end of the DCT that allow types, which would preclude clear-cut basis of a difference in morphology, lo- unambiguous dissection of a uniform clustering into three distinct groups of cation, and function.30 The transition DCT epithelium (Figure 2).39,40 Rodents, cells. The S3 segment is found chieflyin between these cell types occurs in the however, exhibit a transition region41 that the outer stripe of the outer medulla, al- outer stripe of the outer medulla. Near includes cells expressing markers nor- lowing it to be separated from S1 and the end of the TAL, the CTAL comes into mally attributed to downstream seg- S2 simply by selecting the outer stripe contact with its own glomerulus. At this ments, such as the three subunits of as starting material for single-cell isola- point, special epithelial cells called mac- the epithelial sodium channel (ENaC) tion.