Targeted single-cell RNA-seq identifies minority cell types of kidney distal nephron that regulate blood pressure and calcium balance

TL;DR: This work coupled a FACS-enrichment protocol with single-cell RNA-seq analysis to profile the transcriptomes of 9099 cells from the nephron region adjacent to the macula densa and creates a web-based data resource for these cells.

Abstract: A major objective in modern biology is generation of comprehensive atlases of various organs identifying all cell types and their expressed genes. In kidney, extensive data exists for proximal tubule and collecting duct cells, but not for non-abundant intermediate epithelial cell types. Here, we coupled a FACS-enrichment protocol with single-cell RNA-seq analysis to profile the transcriptomes of 9099 cells from the nephron region adjacent to the macula densa. Clusters containing Slc12a3+/Pvalb+ and Slc12a3+/Pvalb- cells were identified as DCT1 and DCT2 cells. The DCT1 cells appear to be heterogeneously associated with variable expression of Slc8a1, Calb1, and Ckb among other mRNAs. No DCT2-specific transcripts were found. The analysis also identified two distinct cell types in the Slc12a1+ portion of Henle’s loop as well as Nos1+/Avpr1a+ macula densa cells. Thus, we identify unexpected cell diversity in the intermediate region of the nephron and create a web-based data resource for these cells.

Summary

INTRODUCTION

• The mammalian kidneys play a crucial role in regulation of body fluid composition and blood pressure.
• BioRxiv preprint 4 cells have been indicative of variable expression along the DCT with detectable levels only in the distal-most DCT14, 15.
• The progressive development and improvement of RNA-seq for comprehensive quantification of gene expression, especially small-sample RNA-seq in microdissected renal tubules17, and more recent single-cell RNA-seq techniques in kidney18, 19, 20, 21, 22, 23, 24, 25, have provided a deep analysis of gene expression in the kidney epithelial cells and have broadened their insights into renal cell identities and functions.

RESULTS

• To provide appropriate context for single-cell RNA-seq (scRNA-seq) analysis of cells in nephron region from cortical thick ascending limb of Henle (CTAL) to the distal convoluted tubule (DCT) to the connecting tubule (CNT) in mouse, the authors began by mapping transcriptomes in microdissected CTAL, DCT, and CNT tubule segments.
• To characterize gene expression in CTAL, DCT, and CNT plus all other renal tubule segments, the authors performed renal tubule microdissection under a standard stereomicroscope (Fig. 1b) followed by small-sample RNA-seq (Supplementary Data 1).
• The copyright holder for this preprint (whichthis version posted July 19, 2020.
• In DCT, some transcripts with no prior known functional roles in the DCT were identified.
• The transcriptomes of each cell cluster are provided in Supplementary Data 7.

Discussion

• A complete understanding of the cellular composition and transcriptional profiles of the cells in DCT has been lacking.
• The authors observed that DCT1 cells are heterogeneous.
• The copyright holder for this preprint (whichthis version posted July 19, 2020.
• A striking observation from the present study is the identification of two CTAL subtypes based on gene expression that might correspond to the morphologically identified cell types, namely cells corresponding to the C5 cluster (Foxq1+Cldn10-Ptger3-) and the C6 cluster (Foxq1Cldn10+Ptger3+).
• In summary, systematic analyses of cellular composition and gene expression provide insights into kidney functions and diseases.

Methods

• Adult mouse kidney cell isolation and single-cell RNA-seq.
• The final cell pellet was resuspended in 30ml FACS buffer (perfusion buffer with 0.05% BSA) and then passed sequentially through 100 µm, 70 µm and 40 µm cell strainers (VWR) to obtain a suspension of isolated cells (single-cell suspension).
• The copyright holder for this preprint (whichthis version posted July 19, 2020.
• The source data underlying Figs. 2a,d, 3a-b, 4a and Supplementary Figs.

Acknowledgments

• The work was primarily funded by the Division of Intramural Research, National Heart, Lung, and Blood Institute (project ZIA-HL001285 and ZIA-HL006129, M.A.K.).
• Confocal images were taken in the NHLBI Confocal Microscopy Core Facility (Christian Combs, Director).
• Cell sorting was performed in the NHLBI Flow Cytometry Core (J. Philip McCoy, Director).
• Tissue sections were prepared in the NHBLI Pathology Facility (Zu-Xi Yu, Director). was not certified by peer review) is the author/funder.
• The copyright holder for this preprint (whichthis version posted July 19, 2020.

Author contributions

• Was not certified by peer review) is the author/funder.
• The copyright holder for this preprint (whichthis version posted July 19, 2020.

Figure Legends

• Overview of renal tubule cell nomenclature and experimental design.
• Fig. 2. Gene expression pattern among mouse renal tubule segments.
• The copyright holder for this preprint (whichthis version posted July 19, 2020.
• The same color codes were used for exons in the splice graph and UCSC Genes Track.
• Heterogeneity of distal convoluted tubule cells revealed by single-cell RNA-seq.

Full text

1
Targeted single-cell RNA-seq identifies minority cell types of kidney distal nephron that
regulate blood pressure and calcium balance
Lihe Chen
1
, Chun-Lin Chou
1
, and Mark A. Knepper
1*
1
Epithelial Systems Biology Laboratory, Systems Biology Center, National Heart, Lung, and Blood
Institute, National Institutes of Health, Bethesda, Maryland
Running title: Single-cell RNA-seq Reveals Transcriptomes of DCT1, DCT2, and Subtypes of CTAL
Cells
*Correspondence and Lead Contact: Mark A. Knepper, MD, PhD, Senior Investigator, Division of
Intramural Research, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892-1603
Email: knepperm@nhlbi.nih.gov
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted July 19, 2020. ; https://doi.org/10.1101/2020.07.19.209627doi: bioRxiv preprint

2
ABSTRACT
A major objective in modern biology is generation of comprehensive atlases of various
organs identifying all cell types and their expressed genes. In kidney, extensive data exists for
proximal tubule and collecting duct cells, but not for non-abundant intermediate epithelial cell
types. Here, we coupled a FACS-enrichment protocol with single-cell RNA-seq analysis to profile
the transcriptomes of 9099 cells from the nephron region adjacent to the macula densa.
Clusters containing Slc12a3
+
/Pvalb
+
and Slc12a3
+
/Pvalb
-
cells were identified as DCT1 and DCT2
cells. The DCT1 cells appear to be heterogeneously associated with variable expression of
Slc8a1, Calb1, and Ckb among other mRNAs. No DCT2-specific transcripts were found. The
analysis also identified two distinct cell types in the Slc12a1
+
portion of Henle’s loop as well as
Nos1
+
/Avpr1a
+
macula densa cells. Thus, we identify unexpected cell diversity in the
intermediate region of the nephron and create a web-based data resource for these cells.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted July 19, 2020. ; https://doi.org/10.1101/2020.07.19.209627doi: bioRxiv preprint

3
INTRODUCTION
The mammalian kidneys play a crucial role in regulation of body fluid composition and
blood pressure. These functions are largely achieved by control of sodium reabsorption across
the epithelia of renal tubules
1, 2
. There are at least 14 different renal tubule segments, each
with characteristic cell types with distinct gene expression profiles and function
3, 4
. Of particular
importance is the distal convoluted tubule (DCT), a short segment that mediates fine regulation
of sodium ion transport
5
. The apical component of sodium transport across the DCT occurs via
the Na
+
-Cl
-
cotransporter NCC (Slc12a3), which is exclusively expressed in DCT cells and the
chief target of thiazide diuretics
6
, a staple in the treatment of many forms of arterial
hypertension. The DCT is believed to be heterogeneous and is separated into at least two
subsegments, DCT1 and DCT2
7, 8, 9
. Both express Slc12a3. DCT1 but not DCT2 expresses the
Ca
2+
-binding protein, parvalbumin (Pvalb). DCT2, in contrast to DCT1, expresses the epithelial
sodium channel (ENaC)
9
. More recent observations indicate that the DCT2 also expresses the
Ca
2+
transporter, Trpv5
10
, and has been proposed to be a site of transepithelial Ca
2+
transport
9,
11
. However, this DCT classification is largely defined based on immunocytochemical results. It
remains unclear how many cell types are in DCT and whether DCT1 and DCT2 are distinct
segments or represent two ends of a continuum.
NaCl transport across the DCT was originally thought to be affected in part by binding of
circulating aldosterone to the mineralocorticoid receptor (MR) in DCT cells
12, 13
. Aldosterone-
MR signaling requires an enzyme 11-β-hydroxysteroid dehydrogenase 2 (Hsd11b2) to
deactivate the more abundant circulating cortisol, which has an equal binding affinity to MR.
However, immunocytochemical and in situ hybridization methods to localize Hsd11b2 in DCT
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted July 19, 2020. ; https://doi.org/10.1101/2020.07.19.209627doi: bioRxiv preprint

4
cells have been indicative of variable expression along the DCT with detectable levels only in
the distal-most DCT
14, 15
. These findings raised doubts about a possible direct role of
aldosterone in the early DCT, and whether the Hsd11b2 positive DCT corresponds to DCT2.
Recent studies further suggest that Slc12a3 (NCC) abundance is regulated by aldosterone only
indirectly via its propensity to cause hypokalemia
5, 16
. This process is thought to be controlled
by multiple receptors, protein kinases (Wnk1, Wnk4, and SPAK), ubiquitin ligase components
(Nedd4l, Klhl3, Cul3), potassium channels, and chloride channels
5, 16
. However, a systematic
mapping of these elements in DCT is currently not available.
The progressive development and improvement of RNA-seq for comprehensive
quantification of gene expression, especially small-sample RNA-seq in microdissected renal
tubules
17
, and more recent single-cell RNA-seq techniques in kidney
18, 19, 20, 21, 22, 23, 24, 25
, have
provided a deep analysis of gene expression in the kidney epithelial cells and have broadened
our insights into renal cell identities and functions. The tubule microdissection method
26
,
however, is unable to effectively isolate the DCT1 and DCT2 due to the overall shortness of DCT
and the lack of a distinct transition point between DCT1 and DCT2. Despite great progress of
comprehensive single-cell RNA-seq in the kidney (‘shotgun’ scRNA-seq), it devotes most of the
sequencing reads to more abundant proximal tubule cells and non-epithelial cells, leading to
analysis of only limited numbers of minority epithelial cell types. Thus, the heterogeneity of cell
types like DCT cells as well as various cell types in the loop of Henle (for example, macula densa
and cortical thick ascending limb cells) are not completely resolved. A solution to this dilemma
is to enrich the target cells before analysis as previously introduced for scRNA-seq analysis of
collecting duct cell types
19
.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted July 19, 2020. ; https://doi.org/10.1101/2020.07.19.209627doi: bioRxiv preprint

5
Here, we describe an enrichment protocol prior to single-cell RNA-seq analysis and
specially focused on the mouse nephron region spanning from the cortical thick ascending limb
of Henle (CTAL) to the DCT. In parallel, we use small-sample RNA-seq to characterize the gene
expression in all 14 renal tubules microdissected from mouse kidneys. We also provide web-
based resources that allow users to explore and download data for future studies.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted July 19, 2020. ; https://doi.org/10.1101/2020.07.19.209627doi: bioRxiv preprint