A fate-mapping approach reveals the composite origin of the connecting tubule and alerts on “single-cell”-specific KO model of the distal nephron

The distal nephron is a heterogeneous part of the nephron composed by six different cell types, forming the epithelium of the distal convoluted (DCT), connecting, and collecting duct. To dissect the function of these cells, knockout models specific for their unique cell marker have been created. However, since this part of the nephron develops at the border between the ureteric bud and the metanephric mesenchyme, the specificity of the single cell markers has been recently questioned. Here, by mapping the fate of the aquaporin 2 (AQP2) and Na+-Cl−cotransporter (NCC)-positive cells using transgenic mouse lines expressing the yellow fluorescent protein fluorescent marker, we showed that the origin of the distal nephron is extremely composite. Indeed, AQP2-expressing precursor results give rise not only to the principal cells, but also to some of the A- and B-type intercalated cells and even to cells of the DCT. On the other hand, some principal cells and B-type intercalated cells can develop from NCC-expressing precursors. In conclusion, these results demonstrate that the origin of different cell types in the distal nephron is not as clearly defined as originally thought. Importantly, they highlight the fact that knocking out a gene encoding for a selective functional marker in the adult does not guarantee cell specificity during the overall kidney development. Tools allowing not only cell-specific but also time-controlled recombination will be useful in this sense.

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Expression of acid-base-related proteins in mesonephric kidney of the rabbit

AJP Renal Physiology ◽

10.1152/ajprenal.1994.267.6.f987 ◽

1994 ◽

Vol 267 (6) ◽

pp. F987-F997 ◽

Cited By ~ 1

Author(s):

T. Matsumoto ◽

C. A. Winkler ◽

L. P. Brion ◽

G. J. Schwartz

Keyword(s):

Collecting Duct ◽

Cell Types ◽

Intercalated Cells ◽

Acid Base ◽

Principal Cells ◽

Collecting Tubule ◽

Metanephric Kidney ◽

Collecting Tubules ◽

Related Proteins ◽

Different Cell Types

The mesonephric kidney, precursor to the metanephric kidney, comprises 30-50 nephrons, each with a glomerulus and proximal, distal, and collecting tubules. Although two different cell types have been identified in the mesonephric collecting tubule, no relationship to cells of the metanephric collecting duct has been established. To characterize expression of some of the acid-base-related proteins, we assayed for carbonic anhydrase (CA) activity and performed immunocytochemistry in mesonephroi from 15- to 20-day-old fetal rabbits. From total RNA, we detected expression of CA II and CA IV mRNA. Microdissected proximal and collecting tubules abundantly expressed both CA II and CA IV, at least to the extent observed in mature metanephric proximal tubules and collecting ducts. Histochemistry confirmed the expression of CA activity in these segments; in the collecting tubule, 28% of the collecting tubule cells were CA rich. Most CA-rich cells showed apical H(+)-ATPase and basolateral band 3 anion exchanger staining consistent with the findings in mature H(+)-secreting (alpha) intercalated cells of the metanephric collecting duct. CA-negative cells could be labeled with an antibody that identifies mature metanephric principal cells. Thus the mesonephric collecting tubule has many cells resembling mature alpha-intercalated cells and a majority of cells resembling principal cells. The similarity to the metanephric collecting duct suggests that the lineages of metanephric alpha-intercalated and principal cells may be closely related to those of the mesonephros.

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Endogenous Notch Signaling in Adult Kidneys Maintains Segment-Specific Epithelial Cell Types of the Distal Tubules and Collecting Ducts to Ensure Water Homeostasis

Journal of the American Society of Nephrology ◽

10.1681/asn.2018040440 ◽

2018 ◽

Vol 30 (1) ◽

pp. 110-126 ◽

Cited By ~ 11

Author(s):

Malini Mukherjee ◽

Jennifer deRiso ◽

Karla Otterpohl ◽

Ishara Ratnayake ◽

Divya Kota ◽

...

Keyword(s):

Epithelial Cell ◽

Notch Signaling ◽

Kidney Development ◽

Collecting Duct ◽

Lithium Treatment ◽

Cell Types ◽

Intercalated Cells ◽

Principal Cells ◽

Adult Kidney ◽

Collecting Ducts

BackgroundNotch signaling is required during kidney development for nephron formation and principal cell fate selection within the collecting ducts. Whether Notch signaling is required in the adult kidney to maintain epithelial diversity, or whether its loss can trigger principal cell transdifferentiation (which could explain acquired diabetes insipidus in patients receiving lithium) is unclear.MethodsTo investigate whether loss of Notch signaling can trigger principal cells to lose their identity, we genetically inactivated Notch1 and Notch2, inactivated the Notch signaling target Hes1, or induced expression of a Notch signaling inhibitor in all of the nephron segments and collecting ducts in mice after kidney development. We examined renal function and cell type composition of control littermates and mice with conditional Notch signaling inactivation in adult renal epithelia. In addition, we traced the fate of genetically labeled adult kidney collecting duct principal cells after Hes1 inactivation or lithium treatment.ResultsNotch signaling was required for maintenance of Aqp2-expressing cells in distal nephron and collecting duct segments in adult kidneys. Fate tracing revealed mature principal cells in the inner stripe of the outer medulla converted to intercalated cells after genetic inactivation of Hes1 and, to a lesser extent, lithium treatment. Hes1 ensured repression of Foxi1 to prevent the intercalated cell program from turning on in mature Aqp2+ cell types.ConclusionsNotch signaling viaHes1 regulates maintenance of mature renal epithelial cell states. Loss of Notch signaling or use of lithium can trigger transdifferentiation of mature principal cells to intercalated cells in adult kidneys.

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Cellular localization of type 5 and type 6 ACs in collecting duct and regulation of cAMP synthesis

AJP Renal Physiology ◽

10.1152/ajprenal.2000.279.1.f185 ◽

2000 ◽

Vol 279 (1) ◽

pp. F185-F194 ◽

Cited By ~ 25

Author(s):

Cécile Héliès-Toussaint ◽

Lotfi Aarab ◽

Jean-Marie Gasc ◽

Jean-Marc Verbavatz ◽

Danielle Chabardès

Keyword(s):

Cellular Localization ◽

Collecting Duct ◽

Cell Types ◽

Functional Expression ◽

Intercalated Cells ◽

Principal Cells ◽

Camp Synthesis ◽

Intracellular Ca ◽

Additive Increase ◽

Different Cell Types

The cellular distribution of Ca2+-inhibitable adenylyl cyclase (AC) type 5 and type 6 mRNAs in rat outer medullary collecting duct (OMCD) was performed by in situ hybridization. Kidney sections were also stained with specific antibodies against either collecting duct intercalated cells or principal cells. The localization of type 5 AC in H+-ATPase-, but not aquaporin-3-, positive cells demonstrated that type 5 AC mRNA is expressed only in intercalated cells. In contrast, type 6 AC mRNA was observed in both intercalated and principal cells. In microdissected OMCDs, the simultaneous superfusion of carbachol and PGE2 elicited an additive increase in the intracellular Ca2+ concentration, suggesting that the Ca2+-dependent regulation of these agents occurs in different cell types. Glucagon-dependent cAMP synthesis was inhibited by both a pertussis toxin-sensitive PGE2 pathway (63.7 ± 4.6% inhibition, n = 5) and a Ca2+-dependent carbachol pathway (48.6 ± 3.3%, n = 5). The simultaneous addition of both agents induced a cumulative inhibition of glucagon-dependent cAMP synthesis (78.2 ± 3.3%, n = 5). The results demonstrate a distinct cellular localization of type 5 and type 6 AC mRNAs in OMCD and the functional expression of these Ca2+-inhibitable enzymes in intercalated cells.

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Single-cell RNA sequencing reveals differential cell cycle activity in key cell populations during nephrogenesis

Scientific Reports ◽

10.1038/s41598-021-01790-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Abha S. Bais ◽

Débora M. Cerqueira ◽

Andrew Clugston ◽

Andrew J. Bodnar ◽

Jacqueline Ho ◽

...

Keyword(s):

Cell Cycle ◽

Single Cell ◽

Molecular Mechanisms ◽

Kidney Development ◽

Collecting Duct ◽

Cell Types ◽

Nephron Progenitors ◽

Developing Kidney ◽

Distal Tubules ◽

Cycle Regulation

AbstractThe kidney is a complex organ composed of more than 30 terminally differentiated cell types that all are required to perform its numerous homeostatic functions. Defects in kidney development are a significant cause of chronic kidney disease in children, which can lead to kidney failure that can only be treated by transplant or dialysis. A better understanding of molecular mechanisms that drive kidney development is important for designing strategies to enhance renal repair and regeneration. In this study, we profiled gene expression in the developing mouse kidney at embryonic day 14.5 at single-cell resolution. Consistent with previous studies, clusters with distinct transcriptional signatures clearly identify major compartments and cell types of the developing kidney. Cell cycle activity distinguishes between the “primed” and “self-renewing” sub-populations of nephron progenitors, with increased expression of the cell cycle-related genes Birc5, Cdca3, Smc2 and Smc4 in “primed” nephron progenitors. In addition, augmented expression of cell cycle related genes Birc5, Cks2, Ccnb1, Ccnd1 and Tuba1a/b was detected in immature distal tubules, suggesting cell cycle regulation may be required for early events of nephron patterning and tubular fusion between the distal nephron and collecting duct epithelia.

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Axial heterogeneity of rabbit cortical collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.1991.260.4.f498 ◽

1991 ◽

Vol 260 (4) ◽

pp. F498-F505

Author(s):

C. L. Emmons ◽

K. Matsuzaki ◽

J. B. Stokes ◽

V. L. Schuster

Keyword(s):

Cross Sections ◽

Cell Function ◽

Rate Coefficient ◽

Collecting Duct ◽

Lectin Binding ◽

Cell Types ◽

Intercalated Cells ◽

Cortical Collecting Duct ◽

Principal Cells ◽

Inner Cortex

The rabbit cortical collecting duct (CCD) consists of three major cell types: principal cells transport K+, beta-intercalated cells absorb Cl-, and alpha-intercalated cells secrete H+. We used functional and histological methods to assess axial distribution of these cell types along rabbit CCD. In perfused CCDs, lumen-to-bath Rb+ rate coefficient (an index of principal cell K+ transport) was not different in tubules from outer cortex (1 mm from renal surface) compared with those from inner cortex (2 mm from renal surface), suggesting that principal cell function is homogeneous along the CCD. In contrast, Cl- rate coefficient (a measure of beta-intercalated cell function) was twice as high in CCDs from outer compared with inner cortex, suggesting heterogeneity of beta-intercalated cells along the CCD. To further investigate these regional differences, we fixed and embedded kidneys and identified three cell types in CCD cross sections using carbonic anhydrase staining and peanut lectin binding. Comparing tubule cross sections from outer with those from inner cortex, we found no axial difference in the fraction of cells that were either principal cells (64%) or total (lectin binding and nonlectin binding) intercalated cells (36%). However, the lectin-binding intercalated cell subset was significantly increased in outer compared with inner cortex. We conclude that there is not heterogeneity of principal cells along the rabbit CCD; however, beta-cell number and function are increased in outer CCD. Collecting duct heterogeneity begins within the cortical segment.

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

10.1101/183376 ◽

2017 ◽

Author(s):

Lihe Chen ◽

Jae Wook Lee ◽

Chung-Lin Chou ◽

Anilkumar Nair ◽

Maria Agustina Battistone ◽

...

Keyword(s):

Cell Surface ◽

Single Cell ◽

Collecting Duct ◽

Extracellular Fluid ◽

Cell Types ◽

Fluid Composition ◽

Intercalated Cells ◽

Rna Seq ◽

Cell Type ◽

Renal Collecting Duct

ABSTRACTPrior 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 fluorescence-activated cell sorting (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, viz. c-Kit. Single-cell RNA-Seq using the 1C- 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 both aquapor¡n-2 and either 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 crosstalk 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.SIGNIFICANCE STATEMENTA long-term goal in mammalian biology is to identify the genes expressed in every cell type of the body. In kidney, the expressed genes (“transcriptome”) of all epithelial cell types have already been identified with the exception of the cells that make up the renal collecting duct, responsible for regulation of blood pressure and body fluid composition. Here, a technique called "single-cell RNA-Seq" was used in mouse to identify transcriptomes for the major collecting-duct cell types: type A intercalated cells, type B intercalated cells and principal cells. The information was used to create a publicly-accessible online resource. The data allowed identification of genes that are selectively expressed in each cell type, informative for cell-level understanding of physiology and pathophysiology.

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Cellular distribution of the potassium channel KCNQ1 in normal mouse kidney

AJP Renal Physiology ◽

10.1152/ajprenal.00087.2006 ◽

2007 ◽

Vol 292 (1) ◽

pp. F456-F466 ◽

Cited By ~ 31

Author(s):

Wencui Zheng ◽

Jill W. Verlander ◽

I. Jeanette Lynch ◽

Melanie Cash ◽

Jiahong Shao ◽

...

Keyword(s):

Collecting Duct ◽

Mouse Kidney ◽

Cellular Heterogeneity ◽

Thick Ascending Limb ◽

Intercalated Cells ◽

Distal Nephron ◽

Rt Pcr ◽

Double Labeling ◽

Cytoplasmic Staining ◽

Principal Cells

Mechanisms of K+ secretion and absorption along the collecting duct are not understood fully. Because KCNQ1 participates in K+ secretion within the inner ear and stomach, distribution of KCNQ1 in mouse kidney was studied using Northern and Western analyses, RT-PCR of isolated tubules, and immunohistochemistry. Northern blots demonstrated KCNQ1 transcripts in whole kidney. RT-PCR showed KCNQ1 mRNA in isolated distal convoluted tubule (DCT), connecting segment (CNT), collecting ducts (CD), and glomeruli. Immunoblots of kidney and stomach revealed a ∼75-kDa protein, the expected mobility for KCNQ1. KCNQ1 was detected by immunohistochemistry throughout the distal nephron and CD. Thick ascending limbs exhibited weak basolateral immunolabel. In DCT and CNT cells, immunolabel was intense and basolateral, although KCNQ1 label was stronger in late than in early DCT. Initial collecting tubule and cortical CD KCNQ1 immunolabel was predominantly diffuse, but many cells exhibited discrete apical label. Double-labeling experiments demonstrated that principal cells, type B intercalated cells, and a few type A intercalated cells exhibited distinct apical KCNQ1 immunolabel. In inner medullary CD, principal cells exhibited distinct basolateral KCNQ1 immunolabel, whereas intercalated cells showed diffuse cytoplasmic staining. Thus KCNQ1 protein is widely distributed in mouse distal nephron and CD, with significant axial and cellular heterogeneity in location and intensity. These findings suggest that KCNQ1 has cell-specific roles in renal ion transport and may participate in K+ secretion and/or absorption along the thick ascending limb, DCT, connecting tubule, and CD.

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NH3 permeability of principal cells and intercalated cells measured by confocal fluorescence imaging

AJP Renal Physiology ◽

10.1152/ajprenal.1995.269.4.f545 ◽

1995 ◽

Vol 269 (4) ◽

pp. F545-F550 ◽

Cited By ~ 14

Author(s):

K. P. Yip ◽

I. Kurtz

Keyword(s):

Time Course ◽

Collecting Duct ◽

Cell Types ◽

Cellular Heterogeneity ◽

Apical Surface ◽

Intercalated Cells ◽

Cortical Collecting Duct ◽

Principal Cells ◽

Image Pairs ◽

Confocal Fluorescence Imaging

The cortical collecting duct (CCD) is an important site for NH3 secretion in mammalian nephron. However, given the cellular heterogeneity of this epithelium, the transcellular sites for NH3 secretion are unknown. In the present study, a dual-excitation confocal microscope was designed and optimized to have sufficient temporal resolution to measure the permeability of ammonia (PNH3) across the basolateral and apical membrane of principal cells (PCs) and intercalated cells (ICs) in perfused rabbit CCDs. The rate of cellular NH3 influx was calculated from the time course of increase in intracellular pH (pHi), measured with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein after 20 mM NH4Cl was added to the bath or luminal perfusate. The time course of increase in pHi was calculated from 488/442 image pairs stored at a rate of 4 Hz. The apparent basolateral and apical PNH3 values of PCs were 36 +/- 5 and 113 +/- 11 microns/s, respectively. The values were 5.0 +/- 0.7 and 34 +/- 3 microns/s after membrane folding correction. The apparent basolateral and apical PNH3 values of ICs were 38 +/- 6 and 132 +/- 15 microns/s. Corrected for membrane folding, the values were 9.0 +/- 1.0 and 47 +/- 5 microns/s, respectively. The results demonstrate that the apical surface was more permeable than the basolateral surface in both cell types. In addition, ICs were more permeable to NH3 than PCs across both membranes. The transcellular PNH3 of PCs and ICs were 27.3 and 29.5 microns/s, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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Functional activity of H-K-ATPase in individual cells of OMCD: localization and effect of K+ depletion

AJP Renal Physiology ◽

10.1152/ajprenal.1996.270.1.f116 ◽

1996 ◽

Vol 270 (1) ◽

pp. F116-F122 ◽

Cited By ~ 9

Author(s):

M. Kuwahara ◽

W. J. Fu ◽

F. Marumo

Keyword(s):

Functional Activity ◽

Collecting Duct ◽

Basolateral Membrane ◽

Cell Types ◽

Intercalated Cells ◽

Principal Cells ◽

Before And After ◽

The Difference ◽

K Depletion

Recent studies have indicated the presence of hydrogen-potassium-adenosinetriphosphatase (H-K-ATPase) in the collecting duct. We examined the localization of functional H-K-ATPase activity in individual cells of the outer and inner stripes of outer medullary collecting ducts (OMCDo and OMCDi). Tubules were isolated from control and K(+)-depleted rabbits and perfused in vitro. Intracellular pH (pHi) of principal cells, intercalated cells, and OMCDi cells was monitored by fluorescence ratio imaging using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). An intracellular acid load was induced by NH3/NH4 prepulse in extracellular Na(+)-, K(+)-, and HCO3(-)-free condition, and then 5 mM K+ was added to the lumen or the bath in the presence of Ba2+. Functional activity of H-K-ATPase was estimated by the difference in the rates of pHi recovery before and after K+ addition. In the control condition, luminal addition of K+ significantly increased the pHi recovery rate by 1.6 +/- 0.4 and 1.9 +/- 0.4 x 10(-3) pH units/s in intercalated calls and OMCDi cells, respectively, but not in principal cells. This K(+)-dependent pHi recovery was inhibited by 63% in intercalated cells and 74% in OMCDi cells in the presence of luminal Sch-28080 (10 microM) but was not affected in the presence of luminal bafilomycin-A1 (10 nM). K+ depletion increased the K(+)-dependent pHi recovery to 2.3-fold in intercalated cells and 2.6-fold in OMCDi cells. By contrast, K(+)-dependent pHi recovery was not detected in the basolateral membrane of any cell types in either the control or the K(+)-depleted condition. These results provide functional evidence that H-K-ATPase is distributed in the luminal membrane of intercalated cells and OMCDi cells and that this ATPase is activated by K+ depletion, suggesting the contribution of intercalated cells and OMCDi cells to K+ conservation in rabbit OMCD.

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Single cell RNA sequencing reveals differential cell cycle activity in key cell populations during nephrogenesis

10.1101/2020.09.16.300293 ◽

2020 ◽

Author(s):

Abha S. Bais ◽

Débora M. Cerqueira ◽

Andrew Clugston ◽

Jacqueline Ho ◽

Dennis Kostka

Keyword(s):

Cell Cycle ◽

Single Cell ◽

Molecular Mechanisms ◽

Kidney Development ◽

Collecting Duct ◽

Cell Types ◽

Ureteric Bud ◽

Nephron Progenitors ◽

Developing Kidney ◽

Distal Tubules

ABSTRACTThe kidney is a complex organ composed of more than 30 terminally differentiated cell types that all are required to perform its numerous homeostatic functions. Defects in kidney development are a significant cause of chronic kidney disease in children, which can lead to kidney failure that can only be treated by transplant or dialysis. A better understanding of molecular mechanisms that drive kidney development is important for designing strategies to enhance renal repair and regeneration. In this study, we profiled gene expression in the developing mouse kidney at embryonic day 14.5 at single cell resolution. Consistent with previous studies, clusters with distinct transcriptional signatures clearly identify major compartments and cell types of the developing kidney. Cell cycle activity distinguishes between the “primed” and “self-renewing” sub-populations of nephron progenitors, with increased expression of the cell cycle related genes Birc5, Cdca3, Smc2 and Smc4 in “primed” nephron progenitors. Augmented Birc5 expression was also detected in immature distal tubules and a sub-set of ureteric bud cells, suggesting that Birc5 might be a novel key molecule required for early events of nephron patterning and tubular fusion between the distal nephron and the collecting duct epithelia.

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