Sugano et al. Evolutionarily conserved DCT gene products 1
Comparative transcriptomic analysis identifies
evolutionarily conserved gene products in the vertebrate
renal distal convoluted tubule
Yuya Sugano1,2, Chiara Cianciolo Cosentino1,2, Dominique Loffing-Cueni1,
Stephan C. F. Neuhauss2# and Johannes Loffing1,3#
1Institute of Anatomy, 2Institute of Molecular Life Sciences, University of Zurich,
Zurich, Switzerland, 3Swiss National Center of Competence in Research “Kidney.CH”,
Zurich, Switzerland
#Corresponding authors
Corresponding authors:
Johannes Loffing Stephan Neuhauss
Institute of Anatomy, Institute of Molecular Life Sciences,
University of Zurich University of Zurich
Winterthurerstrasse 190, Winterthurerstrasse 190,
CH-8057 Zurich, Switzerland CH-8057 Zurich, Switzerland
Phone: +41 (0) 44 635 53 20 Phone: +41 (0)44 635 60 40
Fax: + 41 (0) 44 635 57 02 Fax: +41 (0)44 635 68 97
Email: [email protected] Email: [email protected]
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Sugano et al. Evolutionarily conserved DCT gene products 2
Abstract
Understanding the molecular basis of the complex regulatory networks controlling
renal ion transports is of major physiological and clinical importance. In this study,
we aimed to identify evolutionarily conserved critical players in the function of the
renal distal convoluted tubule (DCT) by a comparative transcriptomic approach. We
generated a transgenic zebrafish line with expression of the red fluorescent mCherry
protein under the control of the zebrafish DCT-specific promoter of the thiazide-
sensitive NaCl cotransporter (NCC). The mCherry expression was then used to isolate
from zebrafish mesonephric kidneys the distal late (DL) segments, the equivalent of
the mammalian DCT, for subsequent RNA-seq analysis. We next compared this
zebrafish DL transcriptome to the previously established mouse DCT transcriptome
and identified a subset of gene products significantly enriched in both the teleost DL
and the mammalian DCT, including SLCs and nuclear transcription factors.
Surprisingly, several of the previously described regulators of NCC (e.g. SPAK, KLHL3,
ppp1r1a) in the mouse were not found enriched in the zebrafish DL. Nevertheless, the
zebrafish DL expressed enriched levels of related homologues. Functional
knockdown of one of these genes, ppp1r1b, reduced the phosphorylation of NCC in
the zebrafish pronephros, similar to what was seen previously in knockout mice for
its homologue, Ppp1r1a. The present work is the first report on global gene
expression profiling in a specific nephron portion of the zebrafish kidney, an
increasingly used model system for kidney research. Our study suggests that
comparative analysis of gene expression between phylogenetically distant species
may be an effective approach to identify novel regulators of renal function.
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Sugano et al. Evolutionarily conserved DCT gene products 3
Introduction
The mammalian renal distal convoluted tubule (DCT) plays a critical role in the
regulation of the whole body NaCl balance and hence, the control of blood pressure
[22]. Moreover, the DCT participates in the regulation of renal K+, Ca2+ and Mg2+
excretion [28,34,35]. The thiazide-sensitive sodium chloride cotransporter (NCC) is
the major apical Na+ and Cl- transport pathway in the DCT [23]. The activity of NCC is
regulated by several kinases including WNKs, SPAK, OSR1 and SGK1. Moreover,
several proteins involved in ubiquitylation (e.g. Nedd4-2, Kelch-like 3 and Cullin 3)
were found to regulate NCC either via direct ubiquitylation of the transporter or via
ubiquitylation of signaling molecules involved in the control of NCC activity [13,31].
The relevance of the DCT, NCC and its regulators is evidenced by two hereditary
diseases with mirroring symptoms [3]. In Gitelman syndrome (GS), patients suffer
from renal salt wasting with hypokalemia, hypocalciuria and hypomagnesemia due
to loss-of-function mutations in NCC [12]. By contrast, in Gordon syndrome,
mutations in WNK1, WNK4, Kelch-like 3 (KLHL3) or Cullin 3 increase NCC activity
and lead to familial hyperkalemic hypertension (FHHt) with hypercalciuria and
hypermagnesemia [1,20].
Our previous work on the transcriptome of the mouse DCT revealed that the
expression of many of these NCC regulators is enriched in the DCT [26]. This allowed
us to identify protein phosphatase 1 inhibitor 1 (I1) as a new regulator of NCC,
confirming that transcriptomic analysis is a powerful method to identify relevant
genes for specific functions along the nephron [2,4,27]. However, a challenge of
transcriptomic analysis resides in the large amount of dataset produced by this type
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Sugano et al. Evolutionarily conserved DCT gene products 4
of comprehensive analysis [5]. This renders selection of candidate genes for further
functional analyses usually difficult. Moreover, testing the in vivo relevance of
candidate genes is complicated by the limited availability of genetically modified
mouse models and the long generation time of mice. To circumvent these limitations,
we reasoned that the zebrafish Danio rerio might serve as an advantageous tool. The
zebrafish is an established vertebrate model because of its small adult size, its high
fecundity, the high degree of genomic conservation to humans and the availability of
genetic tools for rapid and sophisticated genetic manipulations [31]. For kidney
research, the zebrafish proofed already to be an excellent system to study
mechanisms of renal development and pathologies including acute kidney injury,
glomerular disease and polycystic kidney degeneration [6,8,25]. What makes the
zebrafish also attractive for studies on the regulation of renal ion transporters is the
fact that almost all of the major renal ion transporters and channels are conserved in
zebrafish and that the orthologues exhibit the same segmental distribution along the
nephron of mammals and the zebrafish [38]. Consistently, the zebrafish possess the
orthologue of the mammalian NCC that is specifically expressed in the distal late (DL)
segment, the corresponding segment of the mammalian DCT.
Now, we hypothesized that also the most relevant genes involved in regulation of the
DCT function are evolutionarily conserved and hence expressed in both mouse and
zebrafish distal tubules. This should allow us to filter down the vast number of DCT-
enriched genes to the most relevant genes and that could be then tested later for their
significance in the zebrafish. To validate this idea, we (i) developed a transgenic
zebrafish with red fluorescent mCherry protein expression in the DL driven by the
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Sugano et al. Evolutionarily conserved DCT gene products 5
promoter of zebrafish slc12a3 encoding NCC, (ii) isolated the DL tubules from the
adult transgenic zebrafish for RNA-seq, (iii) compared the established transcriptome
of the zebrafish DL with the previously obtained transcriptome of the mouse DCT and
(vi) tested the functional significance of one candidate gene by morpholino-based
knockdown in the zebrafish larval pronephros using custom-made antibodies for
zebrafish NCC. We present a proof-of-concept for the use of the cross-species
transcriptomic analysis in isolating candidate genes for novel regulators of renal
function.
Materials and methods
Zebrafish lines and husbandry
Zebrafish were maintained under a 14h/10h light/dark cycle. Fish were bred as
previously described and the embryos were raised at 28 ⁰C in E3 medium [37]. For
microdissection of DL segments, transgenic zebrafish with mCherry expression in the
DL segments were generated through the Tol2 gateway system [18]. The 1kb
fragment of the promoter of slc12a3 was amplified from WIK genomic DNA with
Phusion High-fidelity DNA polymerase (Thermo Scientific). The primers contained
attB4 and attB1 sequences at the 5’ ends of the forward and reverse primers,
respectively. The PCR product was first subcloned into the pDONR-P4P1R vector and
then recombined into the pDestTol2CG2 vector with mCherry middle element and the
3’-polyA element using the Gateway Vector Conversion kit (Invitrogen).
Plasmid DNAs for microinjection were purified using the plasmid purification kit
(Macherey-Nagel, Oensingen, Switzerland). Tol2 transposase mRNA was in vitro
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Sugano et al. Evolutionarily conserved DCT gene products 6
transcribed using mMessage mMachine Sp6 kit (Life Technologies). The expression
construct was co-injected into fertilized embryos at the one-cell stage with
transposase mRNAs. The injected embryos were screened for cmlc2:eGFP marker and
positive embryos were raised to adulthood. F0 founders were outcrossed to wild-type
zebrafish and resulting F1 offsprings were then screened for mCherry expression in
the DLs. Positive F1 adults were subsequently outcrossed to wild-types to obtain the
stable F2 generation.
Whole-mount in situ hybridization
A digoxigenin-labeled RNA probe was prepared by in vitro transcription of the
zebrafish slc12a3 cDNA fragment using the Roche DIG-RNA Labeling Kit (Roche
Diagnostics, Rotkreuz, Switzerland). Zebrafish embryos were fixed in 4 % PFA in PBS
at 4 ⁰C overnight and whole-mount in situ hybridization was performed as previously
described in [33].
DL tubule microdissection
Tubule isolation from the mesonephros was performed according to [9] with some
modifications to the zebrafish adult mesonephros. Approximately, 20 adult
transgenic zebrafish Tg(slc12a3:mCherry) were anaesthetized and the kidneys were
dissected. Isolated kidneys were incubated in 10 mM DTT for 1 h at room
temperature. Kidneys were then washed in 1 X PBS and digested in 5 mg/ml
collagenase (Roche Diagnostics, Rotkreuz, Switzerland) in Hank’s saline at 28.5 ⁰C for
1 h. DL segments were identified by mCherry transgene expression and
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Sugano et al. Evolutionarily conserved DCT gene products 7
microdissected from the rest of the kidneys under the stereomicroscope. mCherry+
DL segments and mCherry- structures were transferred into 1.5 ml Eppendorf tubes
by pippeting for RNA extractions. Three biological replicates were prepared for each
sample group.
RT-PCR
cDNAs were reverse-transcribed from RNAs of mCherry+ and mCherry- samples. PCR
was performed with a primer pair for slc12a3 (5'- TGGCTTGGCTAGAGATTG -3' and
5'- ATTCATGTTTTTGCCTGC - 3') under the following conditions: 5 min at 94 ⁰C and
then 42 cycles of 30 s at 94 ⁰C, 30 s at 56 ⁰C, and 1 min 72 ⁰C, prior to the final
extension of 5 min at 72 ⁰C.
Zebrafish NCC antibodies
Antibodies recognizing total zebrafish NCC and NCC phosphorylated at Thr49 and Thr
62 (corresponding to Thr53 and Thr58 in mice) were raised in rabbits by
immunizations with the following keyhole limpet hemocyanin (KLH)-coupled,
synthetic peptides and phosphopeptides corresponding to amino acid sequences of
zebrafish NCC: total zNCC (aa 290-307: C-ATPQKQARGFFSYRADIF), pT49-zNCC (aa
46-52: C-GYD-phosphoT-LDAP), and pT62 (aa 58-67: C-FYTN-phosphoT-EVFGR).
The N-terminal cysteine was added for coupling of peptides to KLH. The antisera were
affinity-purified against the respective immunogenic peptides. For phospho-site
specific antibodies, the primary purification was followed by additional affinity
purification against the non-phosphorylated peptides. Peptide-synthesis,
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Sugano et al. Evolutionarily conserved DCT gene products 8
immunizations of rabbits, and antibody purifications were custom-made by Pineda
Antibody Services (Berlin, Germany).
Immunoblotting
The human embryonic kidney cell line HEK293 (ATCC, Manassas, Virginia) was
maintained in MEM (life technologies) with 10% FCS (Amimed) and transfected at
70% confluency with mammalian expression vectors (3 ug DNA) encoding for EGFP
(EX-EGFP-M02, Tebu-bio) and zNCC (dslc12a3_pcDNA3.1-C-(k)DYK, (Genscript),
respectively. Two days after transfection, cells were lysed with RIPA Buffer (Pierce)
and processed for immunoblotting according to standard procedures. 50ug of
proteins were loaded per lane and separated by SDS-Page, blotted on nitrocellulose
membrane (Bio-Rad) and subsequently incubated with affinity–purified antibodies
against total zNCC (1:2000), pT49-zNCC (1:1000), and pT62-zNCC (1:200). Binding
of primary antibodies was revealed by incubation with an IRDye 800 conjugated goat-
anti-rabbit IgG (1:10’000, LICOR, Germany) using a LICOR infrared imager (LICOR).
Equal loading and blotting was controlled by incubating the membrane with a
monoclonal antibody against βactin (1:20’000; Sigma-Aldrich) and a respective
IRDye680 labelled secondary antibody goat-anti-mouse IgG (1:10'000; LICOR,
Germany). In a subset of experiments, phosphorylation of NCC was increased by
incubation for 30 minutes of the transfected HEK293 cells in the presence of the
protein phosphatase 1 inhibitor calyculin A (Cell Signaling) at concentration of 20 nM
in the cell culture medium.
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Sugano et al. Evolutionarily conserved DCT gene products 9
Whole-mount immunofluorescence
Zebrafish larvae and adult kidneys were fixed in 4 % PFA for 30 min at room
temperature (RT). Samples were serially dehydrated and stored in 100% MeOH at -
20 ⁰C at least overnight. Samples were treated with acetone for 7 min at -20 ⁰C and
after washes in 1 X PBST (1 X PBS, 0.1 % Tween 20), blocked in the blocking buffer
(1% BSA, 1% DMSO, 0.1% Tween 20, 5% normal goat serum in 1 X PBS) for 4 hours
at RT. Samples were then incubated with the respective primary antibodies in the
blocking buffer at 4 ⁰C overnight. 1:500 dilution was used for t-zNCC, both pT49-zNCC
and pT62-zNCC. The alpha-subunit of the Na-K-ATPase was detected with a mouse
anti-Na-K-ATPase antibody (Developmental Studies Hybridoma Bank, University of
Iowa) diluted by 1:100. Next day, samples were washed in 1 X PBST several times and
subsequently blocked in the blocking buffer for 3 hours at RT. As a secondary
antibodies, goat anti-rabbit IgG Alexa 488 and goat anti-mouse IgG Alexa 568 were
applied in 1 X PBST with a dilutions of 1:500 at 4 ⁰C overnight. Samples were washed
in 1 X PBST and 1 X PBS and stored in glycerol at 4 ⁰C.
RNA-seq analysis
RNAs were extracted from microdissected DL tubules using the RNA extraction kit
(Macherey-Nagel). Adaptor-ligated cDNA libraries were constructed using Ovation
Single Cell RNA-Seq System (NuGEN). The TruSeq SR Cluster Kit v4-cBot-HS or
TruSeq PE Cluster Kit v4-cBot-HS (Illumina, Inc, California, USA) was used for cluster
generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing was
performed on the Illumina HiSeq 2500 paired end at 2 X126 bp or single end 126 bp
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Sugano et al. Evolutionarily conserved DCT gene products 10
using the TruSeq SBS Kit v4-HS (Illumina, Inc, California, USA). The data are deposited
in the Gene Expression Omnibus database of the National Institutes of Health
(https://www.ncbi.nlm.nih.gov/geo/) under the following accession number
(GSE96519). Full lists of gene expression data were generated by GeneVia
Technologies (Tampere, Finland) and were ordered according to general expression
levels (suppl. table 1) and enrichment in DL (suppl. table 2).
Morpholino injection
Morpholino antisense oligonucleotides (GeneTools LLC, Philamath, Oregon, USA)
were designed to target the translation start sites of the zebrafish ppp1r1b (ppp1r1b-
ATG-MO: 5’-GGATCCATAATGCGCTTTCGTCCTC-3’). The standard morpholino (5’-
CCTCTTACCTCAGTTACAATTTATA-3’) was used as a control. The morpholinos (6
ng/nl) were diluted in RNase free water with 0.1% phenol red as an injection tracer
and injected (1 nl solution / per injection) into fertilized embryos at 1-4 cell stages.
Results
First, we isolated an approximately 1 kb upstream sequence of the slc12a3 gene and
found that this promoter fragment drove expression of mCherry reporter transgene
in the DL of the pronephros (Fig. 1A), similar to the DL restricted expression of
endogenous slc12a3 as revealed by whole-mount in situ hybridization (Fig. 1B).
Furthermore, mCherry expression was visible in the distal portion of adult
mesonephric nephrons. Interestingly, in the adult zebrafish kidney, transgene
expression was observed not only in DL but also in collecting ducts (CD), which are
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Sugano et al. Evolutionarily conserved DCT gene products 11
running as rather thick tubules perpendicular to the DL (Fig. 1C). Nevertheless, the
transgene expression in CDs does not reflect NCC abundance as revealed by
immunostaining of isolated tubules. In these isolated tubules, NCC abundance is
restricted to the DL, but absent from the CD (suppl. Fig. 1). During microdissection,
the mCherry+ CDs were easily separated from the mCherry+ DLs due to their distinct
morphology, their larger diameter and their specific localization in the middle of the
mesonephroi. Subsequently, RNAs were extracted both from microdissected
mCherry+ DL segments and mCherry- structures (Fig. 1D). In order to validate the
distal specificity of the mCherry+ fractions, prior to RNA-seq, RT-PCR was first
performed on cDNAs reverse-transcribed from the extracted RNAs using primer pairs
for slc12a3 and podocin. These experiments demonstrated the presence of a PCR
product for slc12a3 in mCherry+ samples while it was absent in mCherry- samples.
The inverse expression pattern was seen for podocin (Fig. 1E), which is a podocyte-
specific but not DL expressed gene [17]. The weak band for podocin in the mCherry+
sample may have resulted from a slight contamination by glomeruli. Next, RNA-seq
analysis was performed on the mCherry+ and mCherry- fractions. Hierarchical
clustering of the samples indicated that all three mCherry+ samples and mCherry-
samples were grouped together (Fig. 1F).
The genes known to be specific in the distal segments in the pronephros, such as
slc12a3 and the chloride channel K (clcnk), were found enriched in mCherry+ samples
while no enrichment was detected for glomerular, proximal tubule, thick ascending
limb, and collecting duct markers, such as podocin, slc20a1a, trpm7, slc12a1 and
myosin6a, respectively (suppl. table 3). The data confirm that our approach allowed a
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Sugano et al. Evolutionarily conserved DCT gene products 12
successful isolation of DL from the zebrafish mesonephroi though they do not fully
exclude a certain contamination by other cell types. We listed the top 181 genes
(adjusted p-value < 0.01) as DL enriched gene products (suppl. table 1). We used p-
value instead of false detection rate (FDR) to set the threshold since, according to the
Zebrafish Model Organism Database (ZFIN), a number of genes with an FDR greater
than 10 % exhibit DL specific expression in the DL of the zebrafish pronephros [36].
Though contamination by other cell types cannot be fully excluded, the collected
evidence suggests that our approach allows a successful isolation of DL from the
zebrafish mesonephroi. Consistent with an efficient isolation of DL segments, the
zebrafish slc12a3 was found among the top 50 of gene products enriched in the
analyzed tubules according to the log2 fold change. Likewise, a non-mammalian and
teleost-specific NCC homologue, slc12a10.3, was highly enriched in the DL. Next, we
compared the DL transcriptome to our previously obtained list of 339 gene products
enriched in the DCT in the mouse kidney and isolated 13 gene products enriched in
both zebrafish DL and mouse DCT including the known DCT gene products slc12a3,
clcnk and barttin (bsnd) (Fig. 2).
To analyze the regulation of zebrafish NCC (zNCC), we developed three antibodies
against total zNCC (t-zNCC) and two phospho-forms of zNCC (pT49-zNCC and pT62-
zNCC). The immunoblot with t-zNCC antibody on lysates from HEK cells expressing
zNCC showed several bands (Fig. 3A). The bands did not appear in preparations from
control cells transfected with EGFP indicating that they represent zNCC. The
immunoblot with pT49-zNCC antibody showed a strong band at about 150 kDa in the
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Sugano et al. Evolutionarily conserved DCT gene products 13
zNCC transfected cells. Although no signal was detected with the pT62-zNCC antibody
under control conditions, when HEK cells were treated with the protein phosphatase
1 inhibitor, Calyculin A, to block dephosphorylation of zNCC, a weak band at about
150 kDa appeared (Fig. 3B). As this band was absent in preparations from
untransfected control cells treated with 20 nM Calyculin A, it likely represents
phospho-zNCC. Whole-mount immunostaining with these antibodies yielded a
staining pattern that overlapped with that of the in situ hybridization staining of zNCC
transcripts (Fig. 3C). Na-K-ATPase was ranked as one of the most enriched gene
products in the RNA-seq data. Consistent with this, whole-mount immunostaining
revealed very high expression levels of the Na-K-ATPase in the DL segment. Also in
mouse, rat and rabbit, the DCT has the highest Na-K-ATPase activity of all nephron
segments [15], underlining the high ion transport capacity of this segment, which is
apparently relevant not only for the mammalian but also for the teleost kidney.
Furthermore, in immunohistochemistry on sections of the zebrafish pronephros,
pT49-zNCC antibody showed a strong immunofluorescent signal on the apical surface
of the pronephros while the Na-K-ATPase localized to the basolateral plasma
membrane, recapitulating the localization patterns of mammalian NCC and Na-K-
ATPase in the DCT (Fig. 3D).
In previous studies on the mouse, we identified the protein phosphatase 1 inhibitor
1 gene, Ppp1r1a, as a DCT-enriched transcript that significantly regulates the
phosphorylation of mouse NCC (26). This prompted us to check if ppp1r1a is also a
gene product enriched in the zebrafish DL. Interestingly, ppp1r1a is not a DL-enriched
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transcript. However, a closely related homologue, namely ppp1r1b, was found among
the list of DL-enriched gene. Consistent with previous results [30], in situ
hybridization confirmed the DL-specific expression of ppp1r1b in the zebrafish
pronephros (Fig. 4A). To determine whether this gene product is involved in
regulation of NCC phosphorylation in the zebrafish DL just like I1 in the mouse
kidney, we performed morpholino knockdown experiments on ppp1r1b. Whole-
mount immunostaining of ppp1r1b morphants with the phospho-antibodies
demonstrated reductions in the staining intensity of phospho-zNCC compared to that
of controls while no apparent difference in staining intensity was detected by in situ
hybridization for the ppp1r1a mRNA transcripts (Fig. 4B).
Discussion
In the present study, we isolated 13 commonly enriched DCT genes between the
zebrafish and the mouse by the comparative transcriptomic approach. In addition to
slc12a3, two other solute carrier family members, slc16a7 and slc5a3b were also
found DL-enriched. Furthermore, we found two transcription factors, transcription
factor AP-2 b (tfap2b) and estrogen related receptor b (esrrb) among the list. Mice
lacking Tfap2b die early postpartum due to polycystic kidney disease with renal
failure, hypercalcemia, hyperphosphatemia and hyperuremia [24]. This indicates a
critical role of this transcription factor for renal development, differentiation, and
function. Unlike to what the name suggests, the Esrrb does not bind estrogens and
therefore presumably does not mediate the stimulatory effects of estrogens on NCC
[7]. However, Esrrb was implicated in the regulation of NKCC2 function [16] and was
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linked to the control of stem cell renewal and differentiation [10], which may
contribute to the remarkable epithelial plasticity of the DCT [14]. Future work will
need to define the roles of these gene products in the DCT.
To our surprise, the zebrafish orthologues for the major regulators of NCC (e.g. WNKs,
SPAK) in mammals are absent in the list of DL-enriched genes [29]. The specific
regulation of NCC through WNKs and SPAK in the DCT may have evolved in higher
vertebrates only. Interestingly, however, the DL does express, with log2 fold changes
higher than 1, the STE20-like kinase b (slkb) and serine/threonine kinase 38b
(stk38b), which are similar to SPAK. Similarly, the orthologue of Kelch-like 3
homologue, which regulates NCC in mammals, is not DL enriched but another
homologue, klhl23, is present in the list. Therefore, it is tempting to speculate that for
the same conserved cellular processes, teleosts and mammals may have deployed
analogous but different genes during the evolution.
Consistent with this idea, we found ppp1r1b, a close homologue of I1, to regulate
phophorylation of NCC in the zebrafish pronephros. Although the orthologue of I1 in
the zebrafish is ppp1r1c, this gene was not listed in the DL-enriched genes. Instead,
ppp1r1b was found specifically expressed in the DL segment of the pronephros, of
which functional knockdowns led to reduced abundance of NCC in the zebrafish
pronephros. In mammalian kidneys, Ppp1r1b encoding for DARPP-32 is highly
expressed in the thick ascending limb (TAL) [11]. NKCC2 is a close homologue of NCC
localized to the TAL. It remains to be determined whether DARPP-32 regulates
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NKCC2 activity in the TAL, analogous to the NCC regulation by I1 in the DCT, but our
data indicates that the regulatory mechanism of ion transporters by protein
phosphatases may have already existed in a common ancestor, from which teleosts
and mammals have descended.
In conclusion, we present a dataset of gene products that are enriched in the zebrafish
DL segment. The dataset per se is an informative and useful resource in
understanding the function of this segment of the zebrafish kidney, an established
model system for human kidney disease. Furthermore, by comparing this dataset to
the corresponding dataset in the mouse kidney, we demonstrated a proof-of-concept
that the cross-species transcriptomic approach between phylogenetically distant
species can aid an identification of evolutionarily conserved potential core players in
nephron segments. With the availability of mammalian segment specific kidney
transcriptomes (e.g. ref. 2, 4, 5, 19, 27) and ease of generation of transgenic zebrafish,
our approach can also be applied to the other nephron segments. Such approaches
would not only help identify critical genes but also give us important insights into the
evolution of the kidney.
Acknowledgment
The authors would like to thank Drs. Jelena Kühn-Georgijevic and Lennart Opitz
(Functional Genomics Center Zurich) for their assistance with RNA-seq and
bioinformatics analysis. Valuable bioinformatics support was also provided by Ville
Kytölä (Genevia Technologies Ltd). We would also like to thank Kerstin Dannenhauer
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and Michèle Heidemeyer for their technical support including an excellent care of the
zebrafish and the careful execution of the HEK293 cell experiments. This work was
supported by the Hartmann Müller Stiftung (to YS), the Forschungskredit from the
Faculty of Medicine at the University of Zurich (to YS), the RiMED Foundation (to
CCC), the Zurich Center for Integrative Human Physiology (to SN, JL), by project
grants (310030_143929/1, 310030_173276) from the Swiss National Science
Foundation (to JL), and the Swiss National Center for Competence in Research
“Kidney.CH” (to JL).
Disclosures
None.
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References
1. Boyden LM, Choi M, Choate KA, Nelson-Williams CJ, Farhi A, Toka HR, Tikhonova IR,
Bjornson R, Mane SM, Colussi G, Lebel M, Gordon RD, Semmekrot BA, Poujol A,
Valimaki MJ, De Ferrari ME, Sanjad SA, Gutkin M, Karet FE, Tucci JR, Stockigt JR,
Keppler-Noreuil KM, Porter CC, Anand SK, Whiteford ML, Davis ID, Dewar SB,
Bettinelli A, Fadrowski JJ, Belsha CW, Hunley TE, Nelson RD, Trachtman H, Cole TR,
Pinsk M, Bockenhauer D, Shenoy M, Vaidyanathan P, Foreman JW, Rasoulpour M,
Thameem F, Al-Shahrouri HZ, Radhakrishnan J, Gharavi AG, Goilav B, and Lifton RP.
Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities.
Nature 482: 98-102, 2011.
2. Chabardes-Garonne D, Mejean A, Aude JC, Cheval L, Di Stefano A,
Gaillard MC, Imbert-Teboul M, Wittner M, Balian C, Anthouard V, Robert C,
Segurens B, Wincker P, Weissenbach J, Doucet A, and Elalouf JM. A
panoramic view of gene expression in the human kidney. Proc Natl Acad Sci U S
A 100: 13710-13715, 2003.
3. Chen D, and Coffman TM. The kidney and hypertension: lessons from mouse
models. Can J Cardiol 28: 305-310, 2012.
4. Cheval L, Pierrat F, Dossat C, Genete M, Imbert-Teboul M, Duong Van
Huyen JP, Poulain J, Wincker P, Weissenbach J, Piquemal D, and Doucet A.
Atlas of gene expression in the mouse kidney: new features of glomerular
parietal cells. Physiol Genomics 43: 161-173, 2011.
5. Cheval L, Pierrat F, Rajerison R, Piquemal D, and Doucet A. Of mice and men:
divergence of gene expression patterns in kidney. PLoS One 7: e46876, 2012.
6. Cianciolo Cosentino C, Skrypnyk NI, Brilli LL, Chiba T, Novitskaya T,
Woods C, West J, Korotchenko VN, McDermott L, Day BW, Davidson AJ,
Harris RC, de Caestecker MP, and Hukriede NA. Histone deacetylase inhibitor
enhances recovery after AKI. J Am Soc Nephrol 24: 943-953, 2013.
7. Divekar SD, Tiek DM, Fernandez A, and Riggins RB. Estrogen-related
receptor beta (ERRbeta) - renaissance receptor or receptor renaissance? Nucl
Recept Signal 14: e002, 2016.
8. Dong L, Pietsch S, and Englert C. Towards an understanding of kidney
diseases associated with WT1 mutations. Kidney Int 88: 684-690, 2015.
9. Drummond IA, and Davidson AJ. Zebrafish kidney development. Methods
Cell Biol 100: 233-260, 2010.
10. Festuccia N, Dubois A, Vandormael-Pournin S, Gallego Tejeda E, Mouren A,
Bessonnard S, Mueller F, Proux C, Cohen-Tannoudji M, and Navarro P. Mitotic binding
of Esrrb marks key regulatory regions of the pluripotency network. Nat Cell Biol 18: 1139-
1148, 2016.
11. Fryckstedt J, Aperia A, Snyder G, and Meister B. Distribution of
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Sugano et al. Evolutionarily conserved DCT gene products 19
dopamine- and cAMP-dependent phosphoprotein (DARPP-32) in the developing
and mature kidney. Kidney Int 44: 495-502, 1993.
12. Glover M, Zuber AM, and O'Shaughnessy KM. Hypertension, dietary salt intake, and
the role of the thiazide-sensitive sodium chloride transporter NCCT. Cardiovasc Ther 29: 68-
76, 2011.
13. Hadchouel J, Ellison DH, and Gamba G. Regulation of Renal Electrolyte Transport by
WNK and SPAK-OSR1 Kinases. Annu Rev Physiol 78: 367-389, 2016.
14. Kaissling B, and Loffing J. Cell growth and cell death in renal distal tubules, associated
with diuretic treatment. Nephrol Dial Transplant 13: 1341-1343, 1998.
15. Katz AI, Doucet A, and Morel F. Na-K-ATPase activity along the rabbit, rat,
and mouse nephron. Am J Physiol 237: F114-120, 1979.
16. Krid H, Dorison A, Salhi A, Cheval L, and Crambert G. Expression profile of nuclear
receptors along male mouse nephron segments reveals a link between ERRbeta and thick
ascending limb function. PLoS One 7: e34223, 2012.
17. Kramer-Zucker AG, Wiessner S, Jensen AM, and Drummond IA. Organization of the
pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM
domain protein Mosaic eyes. Dev Biol 285: 316-329, 2005.
18. Kwan KM, Fujimoto E, Grabher C, Mangum BD, Hardy ME, Campbell DS, Parant JM,
Yost HJ, Kanki JP, and Chien CB. The Tol2kit: a multisite gateway-based construction kit
for Tol2 transposon transgenesis constructs. Dev Dyn 236: 3088-3099, 2007.
19. Lee JW, Chou CL, and Knepper MA. Deep Sequencing in Microdissected Renal Tubules
Identifies Nephron Segment-Specific Transcriptomes. J Am Soc Nephrol 26: 2669-2677,
2015.
20. Louis-Dit-Picard H, Barc J, Trujillano D, Miserey-Lenkei S, Bouatia-Naji N,
Pylypenko O, Beaurain G, Bonnefond A, Sand O, Simian C, Vidal-Petiot E, Soukaseum C,
Mandet C, Broux F, Chabre O, Delahousse M, Esnault V, Fiquet B, Houillier P, Bagnis CI,
Koenig J, Konrad M, Landais P, Mourani C, Niaudet P, Probst V, Thauvin C, Unwin RJ,
Soroka SD, Ehret G, Ossowski S, Caulfield M, International Consortium for Blood P,
Bruneval P, Estivill X, Froguel P, Hadchouel J, Schott JJ, and Jeunemaitre X. KLHL3
mutations cause familial hyperkalemic hypertension by impairing ion transport in the distal
nephron. Nat Genet 44: 456-460, S451-453, 2012.
21. McCampbell KK, Springer KN, and Wingert RA. Atlas of Cellular Dynamics during
Zebrafish Adult Kidney Regeneration. Stem Cells Int 2015: 547636, 2015.
22. McCormick JA, and Ellison DH. Distal convoluted tubule. Compr Physiol 5:
45-98, 2015.
23. Moes AD, van der Lubbe N, Zietse R, Loffing J, and Hoorn EJ. The
sodium chloride cotransporter SLC12A3: new roles in sodium, potassium, and
blood pressure regulation. Pflugers Arch 466: 107-118, 2014.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Sugano et al. Evolutionarily conserved DCT gene products 20
24. Moser M, Dahmen S, Kluge R, Grone H, Dahmen J, Kunz D, Schorle H,
and Buettner R. Terminal renal failure in mice lacking transcription factor AP-2
beta. Lab Invest 83: 571-578, 2003.
25. Obara T, Mangos S, Liu Y, Zhao J, Wiessner S, Kramer-Zucker AG, Olale F, Schier AF,
and Drummond IA. Polycystin-2 immunolocalization and function in zebrafish. J Am Soc
Nephrol 17: 2706-2718, 2006.
26. Picard N, Trompf K, Yang CL, Miller RL, Carrel M, Loffing-Cueni D, Fenton RA,
Ellison DH, and Loffing J. Protein phosphatase 1 inhibitor-1 deficiency reduces
phosphorylation of renal NaCl cotransporter and causes arterial hypotension. J Am Soc
Nephrol 25: 511-522, 2014.
27. Pradervand S, Zuber Mercier A, Centeno G, Bonny O, and Firsov D. A
comprehensive analysis of gene expression profiles in distal parts of the mouse
renal tubule. Pflugers Arch 460: 925-952, 2010.
28. Rengarajan S, Lee DH, Oh YT, Delpire E, Youn JH, and McDonough AA.
Increasing plasma [K+] by intravenous potassium infusion reduces NCC
phosphorylation and drives kaliuresis and natriuresis. Am J Physiol Renal Physiol
306: F1059-1068, 2014.
29. Richardson C, and Alessi DR. The regulation of salt transport and blood pressure by
the WNK-SPAK/OSR1 signalling pathway. J Cell Sci 121: 3293-3304, 2008.
30. Robra L, and Thirumalai V. The Intracellular Signaling Molecule Darpp-32 Is a Marker
for Principal Neurons in the Cerebellum and Cerebellum-Like Circuits of Zebrafish. Front
Neuroanat 10: 81, 2016.
31. Santoriello C, and Zon LI. Hooked! Modeling human disease in zebrafish. J
Clin Invest 122: 2337-2343, 2012.
32. Sohara E, and Uchida S. Kelch-like 3/Cullin 3 ubiquitin ligase complex and WNK
signaling in salt-sensitive hypertension and electrolyte disorder. Nephrol Dial Transplant
31: 1417-1424, 2016.
33. Sugano Y, Lindenmeyer MT, Auberger I, Ziegler U, Segerer S, Cohen CD, Neuhauss
SC, and Loffing J. The Rho-GTPase binding protein IQGAP2 is required for the glomerular
filtration barrier. Kidney Int 88: 1047-1056, 2015.
34. Terker AS, Zhang C, McCormick JA, Lazelle RA, Zhang C, Meermeier NP, Siler DA,
Park HJ, Fu Y, Cohen DM, Weinstein AM, Wang WH, Yang CL, and Ellison DH. Potassium
modulates electrolyte balance and blood pressure through effects on distal cell voltage and
chloride. Cell Metab 21: 39-50, 2015.
35. Thebault S, Hoenderop JG, and Bindels RJ. Epithelial Ca2+ and Mg2+
channels in kidney disease. Adv Chronic Kidney Dis 13: 110-117, 2006.
1
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3
4
5
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59
60
61
62
63
64
65
Sugano et al. Evolutionarily conserved DCT gene products 21
36. Thisse, B., Thisse, C. (2004) Fast Release Clones: A High Throughput Expression
Analysis. ZFIN Direct Data Submission http://zfin.org/
37. Westerfield M. The zebrafish book : a guide for the laboratory use of zebrafish
(Brachydanio rerio). Eugene, OR: M. Westerfield, 1993.
38. Wingert RA, Selleck R, Yu J, Song HD, Chen Z, Song A, Zhou Y, Thisse
B, Thisse C, McMahon AP, and Davidson AJ. The cdx genes and retinoic acid
control the positioning and segmentation of the zebrafish pronephros. PLoS
Genet 3: 1922-1938, 2007.
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Figure captions
Fig. 1. (A) mRNA expression of the zebrafish slc12a3 in the distal late (DL) of the
pronephros at 48 hpf. (B) Stable expression of mCherry in the zebrafish DL driven by
the slc12a3 promoter in Tg(slc12a3:mCherry) fish at 48 hpf. (C) Ventral view of the
mesonephric kidney from a 20 months old Tg(slc12a3:mCherry) fish. mCherry
positive distal nephrons (arrows) drain into the large collecting duct (arrowheads).
(D) Representative mCherry-positive (+) DL segments and mCherry-negative (-)
nephron segments isolated by free-hand microdissection. (E) A representative RT-
PCR of slc12a3 and podocin mRNAs from mCherry-positive DL and negative segments
demonstrates a strong enrichment of slc12a3 in the mCherry (+) tubules while
podocin is detectable in these samples. (F) Hierarchical clustering depicts the high
consistency of expression patterns for the 3 different samples for each mCherry (+)
and mCherry (-) sample group. Scale bars = 250 μm.
Fig. 2. Venn-diagram showing the number of zebrafish DL enriched gene products and
mouse DCT enriched gene products. The overlap of the circles indicates the gene
products enriched in zebrafish DL and mouse DCT. Gene names, rate of enrichment
and adjusted p values are given in the table.
Fig. 3. Characterization of the affinity-purified anti-zNCC antibodies by
immunoblotting and immunofluorescence. (A) In lysates from HEK cells transfected
with zNCC, anti-t-zNCC antibody detects several bands that are absent in cell
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Sugano et al. Evolutionarily conserved DCT gene products 23
homogenates from HEK cells transfected with EGFP only. pT49-zNCC antibody
detects a strong single band in zNCC transfected cell lysates. Detection of actin serves
to confirm equal protein loading. (B) The pT62-zNCC antibody detects several bands
in lysates from cells transfected with zNCC and treated with 20 nM Calyculin A,
including the band corresponding to the single band detected by pT49-zNCC
(arrowhead). The bands are absent in control cell lysates with the exception of one
band at about 130 kDa. (C) Immunofluorescent detection of total (t-zNCC) and
phospho-zNCC (pT49-zNCC and pT62-zNCC) in the DL of the pronephros at 48 hpf
and detection of the Na-K-ATPase in the pronephros at 30 hpf in whole-mount
preparations of zebrafish larvae. Scale bar = 500 μm. (D) Immunostaining reveals
apical localization of phospho-zNCC (red fluorescence) and basolateral localization of
Na-K-ATPase (green fluorescence) in cryotome-made cross-sections through the DL
of the zebrafish larvae at 72 hpf. Scale bar = 20 μm.
Fig. 4. (A) mRNA expression of the zebrafish ppp1r1b in the DL of the pronephros at
48 hpf. (B) Representative immunostainings and in situ hybridizations of control
zebrafish larvae and larvae with morpholino-based knockdown of ppp1r1b. The
knockdown of ppp1r1b decreases the phosphorylation of zNCC as revealed by
immunostainings with the pT49-zNCC and pT62-zNCC antibodies at 72 hpf, while
mRNA expression of slc12a3 is similar for ppp1r1b morphants and control larvae at
48 hpf. Scale bars = 500 μm.
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