Am J Physiol Regul Integr Comp Physiol 307: R1381–R1395, 2014.
First published October 22, 2014; doi:10.1152/ajpregu.00033.2014.
Review
Morphological and functional characteristics of the kidney of cartilaginous
fishes: with special reference to urea reabsorption
Susumu Hyodo, Keigo Kakumura, Wataru Takagi, Kumi Hasegawa, and Yoko Yamaguchi
Laboratory of Physiology, Atmosphere and Ocean Research Institute, University of Tokyo, Kawshiwa, Chiba, Japan
Submitted 24 January 2014; accepted in final form 17 October 2014
cartilaginous fish; kidney; osmoregulation; transporters; urea
BODY FLUID REGULATION is essential for all organisms to survive
in their respective habitats, including freshwater (FW), seawater (SW), and terrestrial environments. It is well known that
teleost fish regulate their plasma Na⫹ and Cl⫺ concentrations
and osmolality at levels about one-third of SW. Meanwhile,
cartilaginous fishes (sharks, skates, rays, and chimaeras) have
adopted a unique osmoregulatory strategy for adaptation to the
high-salinity marine environment. Cartilaginous fishes control
plasma ions to levels approximately one-half of surrounding
SW while they store high concentrations of the nitrogenous
compound urea as an osmolyte (the so-called “ureosmotic
strategy”). As a result, their body fluids are slightly hyperosmotic to surrounding SW (for instance, plasma osmolality of
houndshark is 1,000 mosmol/kg H2O in SW of 988 mosmol/kg
H2O; Ref. 53), and thus cartilaginous fishes do not typically
suffer dehydration even in the high-osmolality SW environment. To maintain high concentration of urea in the body, urea
production by the ornithine urea cycle (OUC) is essential.
Investigations on the OUC enzymes have proved that the liver
is the primary organ for urea synthesis (2, 3). Furthermore,
Address for reprint requests and other correspondence: S. Hyodo, Laboratory of Physiology, Atmosphere and Ocean Research Institute, University of
Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan (e-mail: hyodo
@aori.u-tokyo.ac.jp).
http://www.ajpregu.org
significant contributions of extrahepatic organs to urea synthesis, such as the muscle and stomach, has also been revealed
(58, 106, 110, 112).
Another essential mechanism to maintain the high internal
urea level is “urea retention.” The kidney is one of the most
important organs for urea retention in cartilaginous fishes,
where urea, as well as water and other small molecules, are
freely filtered by the glomerulus. As mentioned in other review
articles (87, 91), mammals and birds have developed elaborate
mechanisms for renal water reabsorption. Their kidneys contain juxtamedullary nephrons that are characterized by a countercurrent multiplier system. This countercurrent multiplier
system generates an osmotic gradient from the renal cortex to
the inner medulla that enables urine concentration and minimizes water loss from the body. This is a necessary functional
adaptation to survive in terrestrial habitats separated from
water. Similarly, to minimize urea loss to the environment,
cartilaginous fishes must have developed specialized nephron
systems by which urea can be recovered efficiently from the
filtrate. Indeed, the cartilaginous fish nephron can reabsorb
more than 90% of filtered urea from the primary urine (13, 61).
Renal urea excretion thus accounts for only 4 –20% of total
urea loss (93, 126). After transfer from full-strength SW to
diluted SW, the plasma urea level is reduced; in lesser spotted
dogfish Scyliorhinus canicula, this reduction is attributed to a
0363-6119/14 Copyright © 2014 the American Physiological Society
R1381
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Hyodo S, Kakumura K, Takagi W, Hasegawa K, Yamaguchi Y. Morphological and
functional characteristics of the kidney of cartilaginous fishes: with special reference to urea
reabsorption. Am J Physiol Regul Integr Comp Physiol 307: R1381–R1395, 2014. First
published October 22, 2014; doi:10.1152/ajpregu.00033.2014.—For adaptation to highsalinity marine environments, cartilaginous fishes (sharks, skates, rays, and chimaeras) adopt a unique urea-based osmoregulation strategy. Their kidneys reabsorb
nearly all filtered urea from the primary urine, and this is an essential component
of urea retention in their body fluid. Anatomical investigations have revealed the
extraordinarily elaborate nephron system in the kidney of cartilaginous fishes, e.g.,
the four-loop configuration of each nephron, the occurrence of distinct sinus and
bundle zones, and the sac-like peritubular sheath in the bundle zone, in which the
nephron segments are arranged in a countercurrent fashion. These anatomical and
morphological characteristics have been considered to be important for urea
reabsorption; however, a mechanism for urea reabsorption is still largely unknown.
This review focuses on recent progress in the identification and mapping of various
pumps, channels, and transporters on the nephron segments in the kidney of
cartilaginous fishes. The molecules include urea transporters, Na⫹/K⫹-ATPase,
Na⫹-K⫹-Cl⫺ cotransporters, and aquaporins, which most probably all contribute to
the urea reabsorption process. Although research is still in progress, a possible
model for urea reabsorption in the kidney of cartilaginous fishes is discussed based
on the anatomical features of nephron segments and vascular systems and on the
results of molecular mapping. The molecular anatomical approach thus provides a
powerful tool for understanding the physiological processes that take place in the
highly elaborate kidney of cartilaginous fishes.
Review
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KIDNEY OF CARTILAGINOUS FISHES
search is still in progress, a possible model for urea reabsorption in the kidney of cartilaginous fishes is discussed based on
the anatomical features of nephron segments and vascular
systems, and on the results of molecular mapping. Whereas
most of the previous cartilaginous fish research has been
performed in elasmobranchs (sharks, rays, and skates), in this
article, we also focus on holocephalan elephant fish (or elephant shark) Callorhinchus milii. The haploid cellular DNA
content of the elephant fish is much smaller than other cartilaginous fish genomes (48), and the genome database has been
opened for public use (http://esharkgenome.imcb.a-star.edu.sg/).
Recently, elephant fish have attracted attention as a model system
for molecular physiological research of cartilaginous fishes (51,
57, 117–119).
Morphological Features of Cartilaginous Fish Kidneys
Because of its remarkable ability to reabsorb urea, the
morphology of cartilaginous fish kidney has attracted attention
over many years. Although microscopic investigations have
been made in numerous elasmobranch species, detailed descriptions of renal nephrons have been reported by two groups
(44, 67– 69, 71). The kidney of cartilaginous fishes is dorsoventrally flattened tissue located in the caudal part of the
abdominal cavity. As shown in Fig. 1A, the kidney consists of
A
C
SZ
BZ
BZ
SZ
BZ
SZ
SZ
BZ
RC
CV
BZ
RC
B
CD
PS
RC
RC
Sinus zone
Bundle zone
RC
RC
Neck segment
Proximal tubule Ia
Proximal tubule Ib
Proximal tubule II
Intermediate segment I
Intermediate segment II
Early distal tubule
Late distal tubule
Collecting tubule
Fig. 1. Anatomical features of the kidney of cartilaginous fish. A: cross-sectional view of the left-half of the kidney of houndshark stained by immunohistochemistry with the antibody raised against urea transporter (UT). Several lobules (separated by broken lines) are shown. Bundle zone (BZ) is observed as a darker
area than sinus zone (SZ) in this picture, because the collecting tubule in the BZ is stained with the UT antibody. B: magnified view. Large renal corpuscles (RC)
are situated along the boundary between sinus and bundle zones. C: schematic drawing of the course of a single nephron. The subdivisions of each tubular part
are indicated by different symbols. The nomenclature of the nephron segments is largely based on Hentschel (44). CD, collecting duct; CV, central vessel; PS,
peritubular sheath. Scale bars, 1 mm (A) and 100 ␮m (B).
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decrease in urea biosynthesis and a concomitant increase in
plasma clearance rates of urea (40). Since the reduced salinity
did not appear to affect gill permeability to urea in the skates
Raja erinacea and Raja radiata and in the lemon shark Negaprion brevirostris (38, 39, 93), the elevated clearance rates
of urea would largely be attributed to an increase in urea loss
at the kidney (for review see Refs. 41 and 42). Conversely, an
increase in plasma urea, attributed to a decrease in plasma
clearance rates, was observed following transfer of lesser
spotted dogfish to 140% SW (40). Morphological studies have
revealed that the renal tubules of marine cartilaginous fishes
are highly elaborate and show unique features compared with
those of other vertebrates (for review see Ref. 70). The unique
anatomical and morphological features have thus been considered to be important for urea reabsorption. However, a mechanism for urea reabsorption is still largely unknown.
In this article, we focus on recent progress in identification
and mapping of various pumps, channels, and transporters on
the nephron segments in the kidney of cartilaginous fishes. The
molecules include urea transporters, Na⫹/K⫹-ATPase (NKA),
Na⫹-K⫹-Cl⫺ cotransporter, and aquaporins, which most probably all contribute to the urea reabsorption process. The molecular mapping approach provides a powerful tool for understanding the physiological processes that take place in the
highly elaborate kidney of cartilaginous fishes. Although re-
Review
KIDNEY OF CARTILAGINOUS FISHES
the capillary system reported by Lacy et al. (71). The central
vessel originates as a few blind-ending branches at the distal
end of the bundle and runs along the entire bundle in close
contact with the third loop and the final segment (46). Finally,
the central vessel merges with the blood sinuses. Further study
is required to resolve the contradictory observations on the
vascular system in the bundle zone.
Based on the morphological features of its epithelial cells,
the single nephron is divided into numerous segments (Fig. 1C)
(44, 68). Although the terminology for nephron segments
varies among researchers and species investigated (see Ref.
70), the basic patterns of segmentation are consistent. In this
review, we basically follow the terminology by Hentschel (44).
In Table 1, nomenclature for nephron segments is compared
among the present paper, Hentschel (44), and Lacy and Reale
(68). The fine structures of tubular epithelial cells in each
segment are well described in the Refs. 44, 68, and 70.
Beginning at the renal corpuscle, the renal tubule has five
major segments: neck, proximal, intermediate, distal, and collecting segments (Fig. 1C).Each segment is further subdivided.
The neck segment is the descending limb of the first loop. The
proximal segment is long and includes the ascending limb of
the first loop in the bundle zone and most part of the second
loop in the sinus zone. Similar to other vertebrate nephrons, the
proximal segments are characterized by a distinct brush border
on the apical, or lumen-facing, surface of epithelial cells.
A brush border is absent from epithelial cells in the next
part, the intermediate and distal segments. According to Lacy
and Reale (70), this part is analogous to the loop of Henle in
the mammalian kidney. This part contains two easily identifiable segments of nephron. One subsegment is the largest tubule
in the bundle zone (the early distal tubule) consisting of large
cuboidal epithelial cells. The epithelial cells of this part have
highly interdigitated lateral borders and a great density of
mitochondria (44). This portion occupies the majority part of
the third loop and is considered to be a diluting segment (31,
43). The other subsegment is the fourth loop in the sinus zone
(the late distal tubule). This portion is composed of low
columnar epithelial cells with numerous mitochondria (44, 68).
The final tubular segment in the bundle zone is referenced as
either the collecting tubule (the present paper and Ref. 44) or
the distal tubule (68) (Table 1).
Figure 1C shows a schematic drawing of the nephron of the
holocephalan elephant fish (or elephant shark). Our morphological examination clearly showed that the basic configuration
of the nephron is consistent between elasmobranchs and holo-
Table 1. Comparison of nomenclature for nephron segments
The Present Paper
Segments
Zone
Hentschel (Ref. 44)
Lacy and Reale (Ref. 68)
Neck segment
Proximal tubule Ia
Proximal tubule Ib
Proximal tubule II
Intermediate segment I
Intermediate segment II
Early distal tubule (EDT)
Late distal tubule (LDT)
Collecting tubule (CT)
Collecting duct (CD)
Sinus to bundle
Bundle
Bundle to Sinus
Sinus
Sinus
Bundle
Bundle
Sinus
Sinus to bundle
Bundle
Neck segment
Proximal tubule Ia
Proximal tubule Ib
Proximal tubule II
Intermediate segment
Neck segment I, II
Proximal segment I, II
Proximal segment III
Proximal segment III, IV
Intermediate segment I
Intermediate segment II, III
Intermediate segment IV, V
Intermediate segment VI
Distal segment I, II
Collecting duct I, II
Early distal tubule
Late distal tubule
Collecting tubule
Collecting duct
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multiple, irregularly shaped lobules. Each lobule is further
separated into two distinct regions, a sinus zone and a bundle
zone (Fig. 1B). Large renal corpuscles (RC in Fig. 1B) are
situated along the boundary between the two zones. The sinus
zone occupies a larger volume in the kidney, wherein renal
tubules are segregated from one another by vascular space or
blood sinuses. In contrast, in the bundle zone renal tubules are
densely packed. The sinus and bundle zones have also been
referred to as mesial and lateral bundle zones, respectively
(44); however, the spatial relationships between the two zones
(mesial and lateral) are not always correct.
The length of a single nephron is long, and the configuration
of renal tubule is extremely complicated, even compared with
the mammalian nephron. The length of the tubule has been
reported to be up to 9 cm in yellownose skate Raja stabuliforis
(86), about 3.3 cm in spiny dogfish (37), about 5 cm (21) or 8
cm (107) in little skate Raja erinacea, and about 2.8 cm in
common stingray Dasyatis pastinaca (36). In the marine elasmobranchs thus far examined, the nephron makes four loops
within a lobule (Fig. 1C). Beginning at a renal corpuscle, the
renal tubule enters into the bundle zone and forms the first
hairpin loop. The tubule then extends into the sinus zone,
where the tubule meanders through the large blood sinuses and
then turns back again into the bundle zone to form the third
loop. The third loop is longer than the first loop, with the tip of
third loop being convoluted at the distal end of the bundle
zone. The tubule then enters into the sinus zone to form the
final, fourth loop. After the fourth turn, the tubule descends
again to the bundle zone, runs in parallel with the first and third
loops, and joins to the collecting duct (Fig. 1C). The collecting
duct runs toward the adjacent bundle and connects between
adjacent lobules.
In the bundle zone, the resulting five tubules of a single
nephron (the descending and ascending limbs of the first and
the third loops, and the final segment) are enclosed in a sac-like
peritubular (or peribundular) sheath, in which the nephron
segments are arranged in parallel as if they formed a countercurrent system. The cells composing the sheath are connected
to each other by tight junctions, suggesting that the sheath acts
as a barrier separating the microenvironment inside the sheath
from the outside (69). Lacy et al. (71) reported the blood
capillary that forms a loop in the sheath. Meanwhile, Hentschel
et al. (46) described a single lymph capillary-like central
vessel. This capillary system is quite different from the mammalian vasa recta, which functions as a countercurrent exchanging system in the renal medulla, and also different from
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Review
R1384
KIDNEY OF CARTILAGINOUS FISHES
Urea Retention in the Kidney: Urea Transporter
The four-loop structure of the nephron and the resulting five
tubules in the bundle zone are considered to play an important
role in urea reabsorption (see Ref. 70). This hypothesis is
supported by the fact that stenohaline freshwater stingrays
Potamotrygon spp. and Himantura signifer do not use urea as
an osmolyte (112–114) and lack such renal countercurrent
segments (70, 88). Still, a precise mechanism for urea reabsorption has not yet been clarified. This is partly due to the
difficulty in applying classical physiological methods to the
kidney of cartilaginous fishes because of its complex architecture. But recent progress in molecular anatomical approaches
helps us to understand the various physiological processes that
are active in the kidney of cartilaginous fishes, as has been
A
B
PII
PII
LDT
LDT
Fig. 2. Morphological features of holocephalan elephant fish kidney stained
with periodic acid-Schiff (PAS). A: bundle zone. Five tubular segments
(arrowheads; delineated by a dotted line) are packed within the peritubular
sheath. B: sinus zone. Thick tubules represent the proximal segment (PII) of
the second loop, while the late distal tubules (LDT) of the fourth loop are
composed of low columnar epithelial cells. The PII and LDT tubules (represented in the light color and the dark color, respectively) are originated from
a single nephron. Note that the LDT is enfolded by the PII segment. Scale bars,
100 ␮m.
achieved for mammalian kidney. Spatial patterns in the distribution of various pumps, channels, and transporters provide
valuable information on the movement of urea, ions, and water
across each nephron segment.
For many years, urea had been thought to cross the cell
plasma membrane relatively freely by simple diffusion. However, in vitro studies revealed that urea permeability across
artificial lipid bilayers was considerably lower than previously
thought (34, 35). This is particularly true for cartilaginous
fishes. The urea efflux across the shark gill is considerably
lower than that across the teleost gill and across the toad
bladder (92, 93). The epithelial cell membrane of shark gill was
shown to have the highest reported cholesterol-to-phospholipid
molar ratio (30), which partly explains the low permeability to
urea and has been considered to be important for urea retention. In other words, a specific machinery is required for urea
transport across the plasma membrane. Among several molecules reported, facilitative urea transporters (UTs) transport
urea most efficiently and play a crucial role in the urinary
concentration mechanism in the mammalian kidney (see Refs.
91 and 96). In this section, we first briefly summarize the
structure, distribution, and function of mammalian UTs (for
review see also Refs. 63, 97, 101) and then review recent
progresses on cartilaginous fish UTs.
Urea transporters in mammalian kidney. The first UT was
identified from the rat kidney by Hediger and colleagues (134).
This protein is now known as the UT-A2 and is encoded by the
slc14a2 gene. Subsequently, Olives et al. (89) isolated a cDNA
encoding a different UT now known as the UT-B. The UT-A
(slc14a2) gene and the UT-B (slc14a1) gene occur in tandem
on chromosome 18 in the mouse, rat, and human and most
probably represent the result of tandem duplication from the
ancestral UT gene (28).
The UT-B gene contains a single UT domain. A recent study
on the X-ray crystal structure of the UT-B revealed that the
UT-B is a homotrimer and each protomer contains a urea
conduction pore with a narrow selectivity filter, which has two
urea binding sites (74). In contrast, the UT-A gene contains
two UT domains in tandem and produces a variety of isoforms
(UT-A1 through UT-A6) via alternative splicing (29, 59, 63,
101, 103) (Fig. 3A). The rat UT-A gene is composed of 24
exons and contains at least two promoters, UT-A␣ and UT-A␤
(6, 26, 63). UT-A␣ is in the 5=-flanking region of UT-A gene
and responsible for UT-A1, UT-A3, and UT-A4 mRNA transcription. These three isoforms share an identical NH2-terminal
domain but are diverse in their COOH-terminal portions (Fig.
3A). On the other hand, UT-A2 is regulated by the UT-A␤
promoter (located in intron 12 in rat, and in intron 13 in
mouse).
The transcripts for urea transporters have been detected in
several tissues; however, for most of the isoforms, the highest
expression was found in the kidney (7, 63). The major UT-A
isoforms expressed in the kidney are UT-A1, UT-A2, and
UT-A3. In the mammalian kidney, both UT-A1 and UT-A3 are
highly expressed in the inner medullary collecting duct
(IMCD), whereas UT-A2 is expressed in the thin descending
limb of the loop of Henle. In the IMCD, urea is reabsorbed by
UT-A1 and UT-A3 from the forming urine, in which urea is
concentrated after the reabsorption of water, NaCl, and other
useful substances. The urea reabsorption by UT-A1 and
UT-A3 is regulated by vasopressin and other factors via PKA-
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cephalan elephant fish. Each nephron makes four turns and
traverses repeatedly between the bundle zone and the sinus
zone. In the bundle zone, the resulting five tubules are arranged
in a countercurrent loop fashion and are wrapped by the
peritubular sheath, so that in cross-sectional view the five
tubules are frequently observed together in close arrangement
(arrowheads in Fig. 2A). Meanwhile, the configuration of
elephant fish nephron in the sinus zone differs from that of
elasmobranchs. In elasmobranch kidneys, the second and
fourth loops run randomly, whereas in the case of elephant fish,
our observations using serial sections demonstrated that the
second and fourth loops of individual nephrons are packed
together, and that the fourth loop is surrounded by the second
loop (Fig. 2B). In this arrangement, the fourth loops of different nephrons are separated from each other. This unique
configuration in elephant fish kidney can be an advantage in
mapping of various transport proteins onto the renal tubule (see
Urea Retention in the Kidney: Urea Transporter and NaCl
Reabsorption and Its Possible Contribution to Urea Reabsorption: NKA and Na⫹-K⫹-2Cl⫺ cotransporter type-2). As already mentioned, a public genome database for elephant fish
can support this effort. Taken together, these features make the
elephant fish an excellent model for the research of cartilaginous fish kidney.
Review
KIDNEY OF CARTILAGINOUS FISHES
A
C
slc14a2
UT-A1
UT-A2
UT-A3
UT-A4
UT-A5
UT-A6
UT-2a
UT-2b
common region
AAVX01419201.1
B
efUT-1a
UT-1
efUT-1b
efUT-2a
UT-2
UT-3
efUT-3
Fig. 3. Schematic representation of UT isoforms. A: mammalian UT-A isoforms. The UT-A (slc14a2) gene contains two UT domains in tandem and
produces a variety of isoforms via alternative splicing. Each UT domain
contains two subdomains (the UT-A thus has four subdomains represented by
distinct boxes). The first and second UT domains generate UT-A3 and UT-A2,
respectively, while UT-A1 contains both UT domains. B: elephant fish (ef) has
three UT genes (UT-1 to UT-3). Two variant forms having distinct NH2terminal intracellular domains were found for UT-1 and UT-2 (efUT1a,
efUT-1b, efUT-2a, and efUT-2b). Filled bars, putative open reading frames;
empty bars, untranslated regions, broken lines, insertions of introns; filled
circles, putative phosphorylation sites. C: two exons encoding NH2-terminal
domains of UT-2a and UT-2b, respectively, were found to be tandemly
arranged on a single genomic fragment (AAVX01419201.1) and interspaced
with an exon, showing that the two variants are derived from an alternative
splicing event. Filled bars, putative open reading frames; empty bars, untranslated regions, broken lines, insertions of introns.
and PKC-mediated pathways (15, 32, 65, 100, 123). This urea
reabsorption is essential to maintain high urea concentration in
the inner medullary interstitium and to set up the intrarenal
osmotic gradient between the cortex and the inner medulla.
The UT-A1 and UT-A3 double knockout mice (UT-A1/3⫺/⫺)
showed a major urinary concentrating defect (24, 25, 27, 76,
101). The UT-A1/3⫺/⫺ mice drink more water and had an
increased urine volume and a reduced maximal urinary concentrating capacity.
UT-A2 is expressed in the thin descending limb of the loop
of Henle (120). Similar to UT-A1 and UT-A3, UT-A2 is also
upregulated by vasopressin (120) and water deprivation (102).
Under normal laboratory conditions, UT-A2 knockout mice
(UT-A2⫺/⫺) did not display a detectable phenotype (115). Only
when mice were fed a low-protein diet and drinking water was
restricted did UT-A2⫺/⫺ mice show a reduced urinary concentrating capacity compared with wild-type mice, implying that
UT-A2 is important for maintaining a high concentration of
urea in the inner medulla when the supply of urea is limited.
UT-A2 was initially thought to mediate urea recycling from the
ascending vasa recta to the thin descending limb (see Refs. 7
and 63). However, based on the analysis of knockout mice, Li
et al. (76) suggested that UT-A2 mediates urea transport from
the lumen of the thin descending limb to the interstitium.
UT-B has a wide tissue distribution. In the kidney, UT-B is
localized in the endothelial cells of descending vasa recta
(129). The UT-B knockout mice (UT-B⫺/⫺) demonstrated
higher urine output than wild-type mice (8, 72, 132). Moreover, the UT-B⫺/⫺ mice also showed reduced abilities in urea
concentration and water reabsorption, indicating “a urea-selective urinary concentrating defect” (132, 133). The urea reabsorbed from IMCD by UT-A1 and UT-A3 most probably
enters into the ascending vasa recta. Then urea is transferred
from the ascending vasa recta to the descending vasa recta via
UT-B by a countercurrent exchange, which helps to preserve
the urea concentration gradient in the inner medulla (76, 132).
Collectively, the entire urea recycling is carried out by the
concerted action of UTs, enabling the kidney to establish the
osmotic gradient from the cortex to the inner medulla for
antidiuretic action (76).
Urea transporters in cartilaginous fishes. Following the
findings of UTs in mammals, phloretin-sensitive facilitated
UTs have been cloned also in nonmammalian vertebrates. In
cartilaginous fishes, a UT showing high homology to mammalian UT-A2 was cloned in spiny dogfish (104). Since this initial
finding, UTs have been found in other elasmobranchs, including the little skate (82), the Atlantic stingray Dasyatis sabina
(54, 55), the Japanese banded houndshark Triakis scyllium
(52), the winter skate Leucoraja ocellata, and the bluntnose
stingray Dasyatic say (56). Compared with mammalian UTs,
UT-A2 has the highest sequence identity to elasmobranch UTs.
UT mRNAs were expressed in various organs including the
brain, liver, and testis, but the highest levels of expression were
found in the kidney. In contrast to that reported for teleost
fishes, expression of UT was not observed in the gills of
cartilaginous fishes. In teleost fishes, two UT genes have been
found; a teleost UT that is the ortholog of mammalian and
elasmobranch UTs is expressed in the gill ionocytes (81, 121),
while a novel-type UT-C is expressed in the eel kidney (80).
Currently, there is no evidence showing that elasmobranchs
have multiple UT genes. Northern blot analysis revealed that,
in addition to the major band (3.8 kb in Atlantic stingray and
2.3 kb in houndshark), shorter and longer transcripts exist in
the elasmobranch kidney. In Atlantic stingray, two UTs
(strUT-1 and strUT-2) translated from five transcripts of different sizes (from 1509 bp to 5179 bp) have been found in the
kidney (55). The authors proposed that the stingray UT gene
contains six exons (or cassettes) with most of the UT protein
being encoded in the first cassette. The strUT-1 and strUT-2
share cassette I but alternatively use cassettes II and III, in
which stop codons exist. These results strongly suggest that the
two UTs found in Atlantic stingray result from the single UT
gene as a consequence of alternative splicing.
On the other hand, three UTs were found by genome
searching of holocephalan elephant fish. Cloning from the
elephant fish kidney clearly showed that the three UTs are
encoded in separate genes, and they were then designated as
efUT-1, efUT-2, and efUT-3 (57). The efUT-1 is orthologous
to known elasmobranch UTs, whereas efUT-2 and efUT-3 are
novel UTs in cartilaginous fishes. Existence of three UTs was
also shown in another holocephalan species, the spotted ratfish
Hydrolagus colliei (4). Furthermore, in elephant fish, two
variants were found for both efUT-1 and efUT-2, of which the
NH2-terminal intracellular domains were distinct between the
variants (Fig. 3B) (57). Database search detected a single
genomic fragment in which the 5= sequences of efUT-2 variants are tandemly arranged and interspaced with an intron (Fig.
3C). This shows that the two variants for efUT-2 are derived
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efUT-2b
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KIDNEY OF CARTILAGINOUS FISHES
UT-1 and UT-2 have most probably diverged in the cartilaginous fish lineage from an ancestral UT gene by a gene duplication event. Therefore, UT-2-like genes are expected to exist
also in other cartilaginous fish species. Although the phylogenetic position of holocephalan UT-3 is not clear, UT-3 may be
the earliest diverged gene in the UT-A/B group (Fig. 4) (4).
Although the transcripts of all three UTs (UT-1, UT-2, and
UT-3) were strongly expressed in the elephant fish kidney, they
showed distinct tissue distribution patterns (57). UT-1 mRNA
was abundant also in the liver and pancreas, whereas an intense
signal of UT-2 mRNA was found in the brain. UT-1 expressed
in the liver probably contributes to the transportation of urea
synthesized by the hepatic ornithine-urea cycle to the general
circulation. On the other hand, UT-3 mRNA expression was
limited to the kidney and the neighboring interrenal tissue.
These results suggest that the UTs produced from the duplicated genes have been functionally diversified.
Distribution, dynamics, and regulation of urea transporters
in the kidney of cartilaginous fishes. Hyodo et al. (52) revealed,
for the first time, that the houndshark UT is exclusively
expressed in the final segment of nephron, the collecting
tubule.This localization pattern was also confirmed for the UT
of bullshark Carcharinus leucas and efUT-1 of elephant fish
Sea urchin UT-like (XM_781359)
Zebra mbuna UT-like (XM_004575539)
94
98
Medaka UT-like (XM_004079740)
100
Teleost UT-C
Pufferfish UT-C (NM_001037990)
Japanese eel UT-C (AB181944)
Elephant fish UT-3 (AB470077)
Catshark UT (JF891417)
100
92
Houndshark UT (AB094993)
78
Spiny dogfish UT (AF257331)
100
60
Chondrichthyes
Atlantic stingray UT (AY277793)
100
Winter skate UT (AY052552)
Elephant fish UT-1a (AB470073)
Elephant fish UT-2a (AB470075)
57
100
54
89
Gulf toadfish UT (AF165893)
Plainfin midshipman UT (JX899381)
71
Lake magadi tilapia UT (AF278537)
100
Pufferfish UT (NM_001032724)
Teleostei
Medaka UT-like (XM_004072624)
100
UT-A/B
Japanese eel UT (AB049726)
100
100
100
Mouse UT-B (BC100570)
Rat UT-B (NM_019346)
Mammalian UT-B
Human UT-B (NM_001128588)
100
100
63
Edible frog UT (Y12784)
Leopard frog UT (JF755890)
Amphibia
Marine toad UT (AB212932)
100
64
100
Western painted turtle UT-like (XM_005305426)
Chinese soft-shelled turtle UT (JN587496)
Reptilia and Aves
Budgerigar UT-like (XM_005145408)
92
87
Human UT-A2 (X96969)
Whale UT (AY061881)
0.1
100
Mammalian UT-A
Mouse UT-A2 (AF367359)
Fig. 4. Molecular phylogenetic tree of vertebrate UTs by using the neighbor-joining method. The purple sea urchin (Strongylocentrotus purpuratus) UT-like
protein was used as outgroup. Accession numbers are shown in parentheses. The numbers shown on the interior nodes are bootstrap percentages.
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from an alternative splicing of exons 1 and 2 of the efUT-2
gene. Although no direct evidence was found for the efUT-1
gene, the conserved exon-intron organization between the
efUT-1 and efUT-2 genes implies that the two variants for
efUT-1 are also derived by alternative splicing. At present, the
functional significance of splicing variants is unknown. However, the longer variants of both efUT-1 and efUT-2 have
additional putative phosphorylation sites in their NH2-terminal
intracellular sequences compared with the shorter variants
(Fig. 3B). Phosphorylation of UT protein has been implicated
in the regulation of urea-transporting activity (12) and membrane accumulation of UT protein (11, 136). The multiple UTs
may be differently regulated and work in harmony for renal
urea reabsorption, just as for mammalian UTs.
Molecular phylogenetic analysis revealed that the reported
vertebrate UTs are classified into two groups (Fig. 4). The first
group contains only teleost UT-C (80), and the second group
includes other nonmammalian UTs and mammalian UT-A and
UT-B (designated as the UT-A/B group) (4, 57). Elephant fish
UT-1 forms a monophyletic group with elasmobranch UTs in
the UT-A/B group, suggesting orthologous relationship to the
elasmobranch UTs. Elephant fish UT-2 is situated outside of
the efUT-1 and elasmobranch UT group. The holocephalan
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KIDNEY OF CARTILAGINOUS FISHES
A
B
Fig. 5. Immunohistochemical localization of UT in the collecting tubule of the
kidney of houndshark. Immunoreaction was visualized with fluorescein-labeled secondary antibody. In full-strength salt water (SW) condition (A), an
intense signal was observed in the apical plasma membrane, while the apical signal
almost disappeared in diluted (30%) SW condition (B). Scale bar, 20 ␮m.
functions as a rate-limiting step for urea reabsorption in the
houndshark kidney. In the mammalian kidney, the apical
plasma membrane of IMCD was found to be the rate-limiting
membrane for overall transepithelial transport of water and
urea (105). Accumulation of the UT-A1 to the apical plasma
membrane is a key step for the regulation of urea transport in
the IMCD (64). These results imply that the regulatory mechanism for urea transport is, at least in part, common between
mammals and cartilaginous fish.
Regulatory mechanism(s) and factor(s) for the cartilaginous
fish UTs have not been understood. In mammals, it is well
known that vasopressin controls the mRNA expression of UTs
and the apical accumulation of UT-A1 via V2-type vasopressin
receptor (12, 14, 15, 64). In the houndshark, secretion of
vasotocin, a nonmammalian ortholog of vasopressin, is enhanced in concentrated SW, whereas it is decreased in lowsalinity conditions (53). Plasma levels of vasotocin showed
highly positive correlations to the apical UT abundance and to
the plasma urea concentration in houndshark (131). Vasotocin
is thus a possible regulator of UT in the kidney of cartilaginous
fishes, as is the case for vasopressin in the mammalian kidney.
So far, mechanisms of renal vasotocin effects have not been
clarified in cartilaginous fishes. Yamaguchi et al. (130) comprehensively searched for vasotocin receptors in elephant fish,
but elephant fish seems to lack an ortholog to the mammalian
V2-type receptor (V2aR). On the other hand, a novel vasotocin
receptor, which is structurally related to the V2-type receptor
but functionally similar to V1-type receptors (named V2bR),
has been found in all nonmammalian jawed vertebrates including elephant fish (130). The V2bRs are expressed at moderate
levels in the kidneys of elephant fish and medaka. The relationship between the novel V2bR and renal function is of great
interest. In lesser spotted dogfish, Wells et al. (125) reported an
antidiuretic action of vasotocin on glomerular function by
using a perfused kidney preparation. Direct action of vasotocin
on tubular urea reabsorption is thus still under debate.
NaCl Reabsorption and Its Possible Contribution to Urea
Reabsorption: NKA and Na⫹-K⫹-2Cl⫺ cotransporter type-2
To date, only a limited number of membrane transporters
have been identified in the kidney of cartilaginous fishes. The
NKA is a basolaterally located pump protein that generates an
electrochemical driving force for subsequent secondarily active
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(Kakumura K, Hyodo S, unpublished data), suggesting that the
exclusive localization of UT in the collecting tubule is common
among cartilaginous fish. Because the facilitative UT is localized on both apical and basolateral membranes of epithelial
cells, urea transport in the collecting tubule could be attributed
to passive permeation. This hypothesis is supported by the
previous anatomical studies (13, 22). Based on the anatomical
relationships among nephron segments examined in spiny
dogfish and little skate, Boylan and colleagues (13, 22) proposed a model for passive urea reabsorption in the elasmobranch kidney.In their model, urea diffuses from the collecting
tubule into the interstitial fluid around it, lowering the urea
concentration of the final urine.
The importance of the collecting tubule for urea reabsorption
is also supported by the results of renal micropuncture and
microdissection studies (22, 98, 99, 107). In spiny dogfish, urea
concentration of fluid obtained from various puncture sites
along the proximal tubule was not significantly different from that
in plasma (98). Deetjen et al. (22) did not find any reabsorption of
urea in the collecting duct system of little skate, indicating that the
tubular fluid flowing into the collecting duct has already reached
its final low concentration with respect to urea.
In vivo osmotic manipulation has also been applied to
investigate urea regulation in cartilaginous fishes. Acclimation
of elasmobranchs to high and low salinity environments results
in an increase and decrease in plasma urea levels, respectively,
to adjust appropriately their plasma osmolality to altered environmental osmolality (see Ref. 53). Morgan and coworkers
(82) demonstrated that, in the little skate, renal tissue osmolality and urea concentration decreased in response to environmental dilution (transfer from 100% SW to 50% SW). At the
same time, the levels of UT transcripts were significantly
diminished in the kidney, implying that the downregulation of
UT mRNA expression led in turn to a decrease in renal tubular
urea reabsorption. On the other hand, Janech et al. (55) detected a significant decrease in relative abundance of the 5.5-kb
transcript of strUT after transfer from 100% SW to 50% SW of
Atlantic stingray but not for other three transcripts (2.8 kb, 3.8
kb, and 4.5 kb). In the houndshark, the abundance of UT
mRNA did not differ among control (100% SW), high (130%
SW), and low (30% SW) salinity groups (131). Therefore,
transcriptional regulation of UT expression is still under debate. In a ureotelic teleost fish, the gulf toadfish (Opsanus
beta), the expression of UT mRNA in the gill is most probably
regulated by endocrine (cortisol) and neural (5-HT) systems
(79). The promoter region of the toadfish UT gene contains
glucocorticoid response element (GRE) half-sites and is activated by cortisol infusion (94).
In parallel with changes in plasma urea levels, salinity
manipulation significantly decreased the abundance of UT
protein in the collecting tubule of the houndshark in low (30%
SW) salinity environment (131). The subcellular UT distribution was also dramatically changed. UT in the apical plasma
membrane of collecting tubule almost completely disappeared
in 30% SW (Fig. 5), whereas it slightly increased in 130% SW
compared with 100% SW group (131). Conversely, reverse
transfer of fish from 30% to 100% SW restored UT in the
apical membrane. A significant positive correlation was found
between the apical UT abundance and plasma urea levels
(131). Therefore, the accumulation of UT in the apical plasma
membrane of the collecting tubule is important and most likely
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KIDNEY OF CARTILAGINOUS FISHES
high urea concentration in the blood of cartilaginous fishes.
This requires that a specific mechanism must exist in the
kidney, which enables “uphill” urea transport from the primary
urine to the circulation. The existence of sodium-coupled
active urea transporting machinery, such as an apical sodium/
urea cotransporter and a basolateral sodium/urea antiporter, has
thus been discussed (122). However, as mentioned above, we
could not find any NKA signal in the collecting tubule where
UT was exclusively localized (52). While the linkage of
sodium reabsorption to the urea reabsorption mechanism is still
unclear, a suggestion derives from knowledge of the unique
vascular element reported by Hentschel et al. (46). They found
a single lymph capillary-like central vessel. As already described in Morphological Features of Cartilaginous Fish Kidney, the central vessel originates as a few blind-ending
branches at the distal end of the tubular bundle, runs in close
contact with the collecting tubule and the third loop, and finally
merges with the large venous sinusoid capillaries of the renal
portal system. Taken together, this morphological result (46)
and the molecular mapping data provide the model for passive
urea reabsorption shown in Fig. 6. In this model 1) massive
reabsorption of NaCl occurs in the early distal tubule (Fig. 6B).
The resulting increase in osmolality of the interstitium causes
absorption of water from an undefined segment in the bundle
zone. Low-urea fluid, in which the urea concentration is considerably lower than that in the blood, is thus generated within
the bundle. 2) The low-urea fluid flows unidirectionally in the
central vessel from the tip of the bundle to the renal portal
system because of high resistance at the peritubular sheath and
low resistance in the direction of the sinus zone (Fig. 6B). 3)
The reabsorption of NaCl, water, and other solutes including
K⫹ and glucose results in an increase in urea concentration in
the forming urine. The urea-rich forming urine passes into the
collecting tubule in which UT is expressed in the apical and
basolateral membranes of the epithelial cells (Fig. 6C). 4)
Reabsorption (or countercurrent exchange) of urea may then
occur from the high-urea fluid in the collecting tubule to the
low-urea fluid in the central vessel via the facilitative UT (Fig.
6D). Further investigations on the localization of other transport proteins can test this hypothetical model.
Urea Synthesis in the Kidney
As has been described in this article, urea reabsorption is the
crucial function in the kidney of cartilaginous fishes. In addition, we recently found mRNA expressions and activities of
ornithine-urea cycle (OUC) enzymes, such as the rate-limiting
carbamoyl phosphate synthetase III (CPSIII), ornithine transcarbamylase (OTC), and arginase in the kidney of holocephalan elephant fish (110), suggesting that the cartilaginous fish
kidney is also involved in urea production. In ureotelic or
ureosmotic fish such as marine cartilaginous fishes, glutamine
is an essential material for urea production (124) since CPSIII
utilizes glutamine, rather than ammonia, as its substrate for
carbamoyl phosphate formation. We found that mRNA expression and enzyme activity of glutamine synthetase (GS) were
the highest in the kidney and were higher than those in the
liver, the major organ for urea production (110).
In situ hybridization was applied to identify the tubular
segments that express the OUC enzyme mRNAs (Fig. 7). We
investigated the distribution of GS and OTC mRNAs in the
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transports through symport or antiport with Na⫹. In the elasmobranch kidney, tubular urea reabsorption is reported to be
correlated with sodium reabsorption at a ratio of 1.6:1, over a
wide range of urine flow rates and urea reabsorption values
(98). This observation led the researchers to expect that NKA
contributes importantly to the urea reabsorption process.
In mammalian nephrons, NKA is abundant in the thick
ascending limb of the loop of Henle, where NaCl is actively
reabsorbed from the forming urine via an apically located
Na⫹-K⫹-2Cl⫺ cotransporter type-2 (NKCC2). The NKCC2 is
a kidney-specific transporter and belongs to the cation-Cl
cotransporter family, which also includes the NKCC1, K-Cl
cotransporters, and Na-Cl cotransporters (33, 78). The active
reabsorption of NaCl dilutes the forming urine passing through
the thick ascending limb of the mammalian nephron (the
so-called diluting segment). In the houndshark kidney, NKAimmunoreactive signal was widely distributed throughout the
nephron. In particular, the convoluted and ascending parts of
the third loop in the bundle zone (the early distal tubule) were
intensely stained using NKA antibody (52). This result is
consistent with the morphological characteristics of this segment; the epithelial cells of the early distal tubule have elaborate basolateral infoldings and numerous large mitochondria
(44, 68). The NKA signal is found also in the second and fourth
loops in the sinus zone, whereas the final collecting tubule is
scarcely stained using the NKA antibody. This is one of the
reasons we concluded that urea is passively reabsorbed in the
collecting tubule by facilitated diffusion through the UT (52).
In accord with the intense NKA staining, the third loop of
the elasmobranch nephron (the early distal tubule) has been
considered to be a diluting segment (31, 43). Microperfusion of
this segment exhibited high rates of Cl⫺ absorption in spiny
dogfish. Furosemide, an inhibitor of NKCC2, but neither
amiloride (an inhibitor of the ENaC epithelial sodium channel
and Na⫹/H⫹ exchangers) nor hydrochlorothiazide (an inhibitor
of the Na⫹-Cl⫺ cotransporter) added to the luminal perfusate
abolished the Cl⫺ absorption (31). Electrophysiological properties of this segment were consistent with the NKCC2-dependent NaCl absorption mechanism and similar to those seen in
the mammalian diluting segment (43). Expression of NKCC2
mRNA was evident in the kidney of spiny dogfish (33);
Biemesderfer et al. (10) further demonstrated NKCC2-like
immunoreactivity on the apical membrane of the third loop.
Taken together, it is most probable that NaCl is actively
reabsorbed when the forming urine passes through the third
loop. In the kidney of spiny dogfish, Biemesderfer et al. (10)
obtained immunoreactive signals from many tubular segments
using the NKCC antibody, suggesting that other segments also
function as diluting segments. This antibody was, however,
raised against the NKCC1 of spiny dogfish. Indeed, Western
blot analysis showed two immunoreactive bands for kidney
proteins. Further studies using homologous probes and/or antibodies are necessary to identify the NKCC2-positive diluting
segment(s). We preliminary detected expression of NKCC2
mRNA in the early and late distal tubules (Kakumura K,
Hyodo S, unpublished data).
Although positive correlation has been observed between
reabsorption of Na⫹ and urea (98), any functional link between
the machinery for sodium reabsorption and that for urea reabsorption has yet to be clarified. In addition, there is a remaining
critical problem for a urea reabsorption mechanism; that is, the
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KIDNEY OF CARTILAGINOUS FISHES
A
B
LDT
LDT
NKA,
NKCC2,
AQP
RC
RC
CV
CV
UT
CT
EDT
NKA, NKCC2
C
D
LDT
LDT
RC
RC
CV
CV
CD
CD
EDT
EDT
elephant fish kidney, because of their high expression levels
among the OUC enzyme mRNAs (110). In situ hybridization
clearly showed that GS and OTC mRNAs were distributed
only in limited tubular segments in the sinus zone (Fig. 7,
A–D). No colocalization was observed with NKCC2 mRNA,
which is expressed in the late distal segment (the fourth loop)
in the sinus zone (Fig. 7, G and H) (Kakumura K, Hyodo S,
unpublished data). Taken together with the morphological
features, the positive tubules for GS and OTC mRNAs are the
proximal segments of the second loop. OTC mRNA signals
were found only in a portion of the second loop and were
colocalized with GS mRNA without exception in the same
tubular segments (Fig. 7, A–D), supporting our notion that the
renal tubule contributes to urea production. On the other hand,
distribution of GS mRNA was much broader in the second loop
than that of OTC mRNA. Therefore, GS in the proximal tubule
is probably involved, in addition to urea production, in further
functions, such as detoxification and recycling of ammonia.
Recently, a growing body of evidence has suggested that
Rhesus (Rh) glycoproteins possess ammonium transporting
ability. Six clusters of the Rh family have been defined by an
extensive molecular phylogenetic analysis (49, 50). In hound-
shark, Rhp2 was found to distribute in the 2nd and 4th loops of
nephron (84). The Rhp2 was localized on the apical membrane
of those segments, implying that Rhp2 facilitates ammonia
reabsorption or secretion. We searched for Rh protein-like
genes from the elephant fish genome database and cloned four
partial mRNAs. Molecular phylogenetic analysis revealed that
the cloned molecules are grouped into known Rhag, Rhcg,
Rh30, and Rhp2 lineages, respectively (Fig. 8). Among them,
only the Rhp2 mRNA was highly expressed in the kidney of
elephant fish, confirming the result in houndshark (Fig. 9).
Expression of Rhag-like mRNA was found in the erythrocytes
and spleen, while Rhcg-like mRNA expression was detected in
the gill. This tissue distribution pattern supports the classification of Rh mRNAs (83, 85, 128). In situ hybridization using
adjacent sections demonstrated that the mRNAs of GS, OTC,
and Rhp2 were colocalized in the same nephron segment of the
proximal tubule (Fig. 7, A–F). These results suggest that, in the
limited segment of the proximal tubule, ammonia is absorbed
either from the sinus blood or from the primary urine to
produce urea. In our preliminary investigation, apical localization of Rhp2 was detected with a heterologous antibody for
houndshark Rhp2. While the result implies the reabsorption of
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CD
CD
EDT
Fig. 6. Schematic diagram showing a urea reabsorption model in the kidney of cartilaginous fish.
A: localization of transporting molecules (indicated in italics). Na⫹/K⫹-ATPase (NKA) and
Na⫹-K⫹-2Cl⫺ cotransporter type-2 (NKCC2) are
expressed in the early distal tubule (EDT; the 3rd
loop) and the late distal tubule (LDT; the 4th
loop), while UT is expressed in the collecting
tubule (CT). Cuttler et al. (20) showed immunoreaction of AQP4 in multiple segments including
the LDT. B: as a first step, NaCl (filled arrow) and
water (open arrow) are reabsorbed from the EDT
(diluting segment) and undefined segment(s), respectively, in the bundle zone. Low-urea fluid
flows unidirectionally in the central vessel to the
renal portal system. C: urea-rich forming urine
passes into the collecting tubule where UT is
expressed. D: reabsorption (or countercurrent exchange) of urea then occurs from the collecting
tubule to the central vessel. CD, collecting duct;
CV, central vessel; RC, renal corpuscle.
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A
GS1
C
D
OTC
E
F
Rhp2-like
H
G
Pufferfish Rhcg1 (AB218981)
100
Pufferfish Rhcg2 (AB218982)
72
Chicken Rhcg (NM_001004370)
79
Frog Rhcg (NM_001003661)
85
Human RhCG (NM_016321)
99
Cattle Rhcg (BC111288)
99
Elephant fish Rhcg-like (AB872670)
91
Pufferfish Rhbg (AB218980)
90
NKCC2
Frog Rhbg (AY619985)
Fig. 7. In situ hybridization of GS1, OTC, Rhp2-like, and NKCC2 mRNAs in
the kidney of elephant fish. Low power micrographs are shown in A, C, E, and
G. Intense signals for OTC and Rhp2 mRNAs were colocalized in the same
tubule (arrows in D and F). These tubules also expressed GS1 mRNA (arrows
in B), but distribution of GS1 mRNA was much broader (filled arrowheads in
B). The GS1, OTC, and Rhp2 mRNAs were not expressed in the fourth loop
(the LDT), in which NKCC2 mRNA was expressed (open arrowheads in B, D,
F, H). Scale bars, 250 ␮m (A, C, E, G) and 50 ␮m (B, D, F, H).
⫹
⫹
⫹
Cattle Rhbg (BC133318)
88
50
Human RhBG (AF193807)
100
Human RhAG (AF237382)
100
80
Cattle Rhag (NM_174171)
Chicken Rhag (NM_204464)
Frog Rhag (AY865610)
99
Pufferfish Rhag (AB218979)
72
66
ammonia from the forming urine, further study using a homologous antibody is required to reach conclusions.
Other Transporting Molecules Expressed in the
Cartilaginous Fish Nephron
Chicken Rhbg (NM_204367)
95
Elephant fish Rhag-like (AB872669)
100
Elephant fish Rhp2-like (AB872668)
99
100
Houndshark Rhp2 (AB287007)
Frog Rhp2 (AY865611)
81
⫹
Urine acidification: H -K -ATPase and Na /H exchangers.
Urine acidification is considered to help to reduce the formation of carbonate precipitates, which is advantageous for excretion of divalent cations in SW fish. In southern flounder
Paralichthys lethostigma, urine pH is slightly lower than blood
⫺
pH (47). Excretion of NH3/NH⫹
4 and HCO3 /CO2 is also
⫹
regulated by H secretion by the renal tubule. Although the
mechanism(s) of urinary acidification in elasmobranchs are
still largely unknown, they produce urine with low pH.
Na⫹/H⫹ exchanger (NHE) and H⫹-K⫹-ATPase are candidates
for excreting H⫹ from the renal tubule.
Pufferfish Rhp2-2 (AY 116073)
Pufferfish Rhp2-1 (AY 116072)
80
Elephant fish Rh30-like (AB872671)
Pufferfish Rh30 (AAM48580)
99
Frog Rh30 (AY865609)
90
Chicken RhD (NM_204467)
79
Human Rh polypeptide (M34015)
93
100
Human RhD (EU557240)
Fruit fly Rh50 (AF172639)
Fig. 8. Molecular phylogenetic tree of vertebrate Rh family proteins by using
the neighbor-joining method. The proteins obtained from elephant fish are
shown in bold. The fruit fly Rh50 (AF172639) was used as outgroup.
Accession numbers are shown in parentheses. The numbers shown on the
interior nodes are bootstrap percentages.
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Kempton (60) showed acidification of perfusate in the proximal and distal thin segments of the nephron in spiny dogfish.
The acidification in the proximal tubule was also found in the
little skate (23). In spiny dogfish, infusion of an inhibitor for
mammalian gastric H⫹-K⫹-ATPase Sch28080 abolished over
80% of basal acid excretion, implying that H⫹-K⫹-ATPasedependent mechanism(s) for urine acidification exist in the
elasmobranch kidney (108). These authors conducted immunohistochemistry using an antibody directed against the
COOH-terminus of porcine gastric H⫹-K⫹-ATPase ␣-subunit
and found that the apical and basolateral membranes of many
nephron segments were stained with the antibody. In particular,
the basolateral membranes of the early distal tubule and the late
distal tubule and the apical membrane of the collecting duct
were intensely stained (108). Hentschel et al. (45) examined in
lesser spotted dogfish, using a different antibody raised against
an extracytoplasmic domain peptide of the rat H⫹-K⫹-ATPase.
They found immunoreaction in the apical membranes of the
proximal segments and the late distal tubule. Both studies thus
revealed immunoreactive H⫹-K⫹-ATPase signals in the proximal and distal tubules, while the localization of signals was
not consistent. The use of an antibody raised against the
homologous protein will be needed.
NHEs belong to the Slc9 family that consists of 13 proteins
in mammals. Among NHEs, NHE3 is highly expressed in the
kidney as well as in the gastrointestinal tract. In the kidney,
B
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KIDNEY OF CARTILAGINOUS FISHES
R1391
0.16
Rhp2-like
Rh30-like
Rhag-like
Rhcg-like
Rh mRNA / ACTB mRNA
0.14
0.12
0.12 ± 0.03
Fig. 9. Tissue distribution of mRNAs encoding Rhp2-like,
Rh30-like, Rhag-like, and Rhcg-like proteins. Data are presented as means ⫾ SE. Values of each mRNA were normalized by the value of beta actin mRNA. Bra, brain; Gil, gill;
Kid, kidney; Liv, liver; Spl, spleen; Int, spiral intestine; Mus,
muscle; Tes, testis; Ova, ovary; Ery, erythrocytes.
0.03
0.02
0.01
Bra
Gil
Kid
Liv
Spl
Int
Mus
Tes
Ova
NHE3 is localized on the apical membranes of the proximal
tubule and the thick ascending limb of the loop of Henle. The
elasmobranch ortholog of mammalian NHE3 was first cloned
from the gill of Atlantic stingray (18). In the gills of elasmobranchs, NHE3 is localized on the apical membrane of ionocytes and is involved in acid excretion, Na⫹ uptake, and Ca2⫹
homeostasis (17, 18, 109). In the houndshark, alternatively
spliced variants have been found in the gill (NHE3g) and the
kidney/intestine (NHE3k/i). The NHE3k/i is expressed on the
apical membranes of a part of the proximal tubule, the early
distal tubule and a part of the late distal tubule (75). The
existence of NHE3 in the proximal tubule is consistent with the
previous result; vesicles of brush-border membrane isolated
from the kidney of spiny dogfish had Na⫹/H⫹ exchange
activity that was inhibited by amiloride (9). Since NHE3 was
coexpressed with NKA in all three segments, it is most likely
that low intracellular Na⫹ concentration caused by NKA is
used as a driving force for exchanging Na⫹ and H⫹ via NHE3.
In addition, vacuolar-type H⫹-ATPase (V-ATPase) was found
on the apical membrane of the proximal tubule and the late
distal tubule, implying that the V-ATPase is also involved in
urine acidification (84).
Water reabsorption: aquaporins. Water reabsorption is one
of the most important functions in tetrapod kidneys. This is
particularly true in birds and mammals, and their kidneys have
developed the urine concentrating mechanism consisting of a
juxtamedullary nephron and countercurrent multiplier and exchange systems (87, 91). As already described in the previous
sections, marine cartilaginous fishes maintain their body fluid
slightly hyperosmotic to the surrounding SW. Therefore, they
are not dehydrated even in the high-osmolality marine environment, and the requirement for water retention is different
from marine teleost fishes and terrestrial vertebrates. Nevertheless, it is highly probable that water reabsorption is a critical
step for urea reabsorption process (see NaCl Reabsorption and
Its Possible Contribution to Urea Reabsorption: NKA and
NKCC2 and Fig. 6).
As reviewed by Nishimura and Yang (87), aquaporins
(AQPs) are a family of small, hydrophobic proteins. AQPs are
phylogenetically old molecules and have been found in most
organisms including plants, microbial organisms, invertebrates, and vertebrates. Thirteen AQPs have been identified in
Ery
mammals, whereas 10 of the 13 AQPs have also been found in
birds (87). It is well recognized that AQP2 is localized in the
apical membrane of the collecting duct and plays an important
role in urine concentration via vasopressin/vasotocin-dependent regulatory signaling. We recently found that the AQP0
paralog, which is considered to be an ancestral molecule of
AQP2, plays an important role in water retention in the kidney
of African lungfish Protopterus annectens during the estivation
period (66). In ray-finned fishes, however, neither AQP2 nor
AQP0 paralog has been found; no research has been conducted
on AQP2 and AQP0 paralog in cartilaginous fishes. Furthermore, among the 13 AQPs, AQP3, 7, 9, and 10 are identified
as aquaglyceroporins and transport not only water but also
small uncharged molecules, such as glycerol and urea (95).
AQP research is thus important for understanding both water
retention and urea retention in the kidney of cartilaginous
fishes.
To date, only AQP4 has been reported in spiny dogfish (19).
The dogfish AQP4 exhibited osmotic water permeability, but
no permeability for urea and glycerol, suggesting the waterselective transport property of AQP4 in spiny dogfish as has
been observed in other vertebrates. The highest expression of
AQP4 was observed in the rectal gland. The elasmobranch
rectal glands secrete NaCl-rich and urea-poor fluid to maintain
the low (relative to seawater) NaCl levels in plasma (90). The
low urea permeability in the isolated membrane of the shark
rectal gland (135) is consistent with the low urea permeability
of AQP4. In the kidney, immunoreactive AQP4 signals were
observed in multiple intermediate and distal segments both in
the sinus zone and the bundle zone; however, further detailed
analysis will be needed to identify membrane localization (20).
Glucose reabsorption. Glucose is the main source of energy
in eukaryotes and is transported across the plasma membrane
by carrier proteins, the glucose transporters. To date, three
families of glucose transporters have been found in the human
genome: 1) the SLC2 family of facilitated diffusion-type transporters (GLUT1 to 14); 2) the SLC5 family of sodium-driven,
secondarily active glucose cotransporters (SGLTs); and 3) the
SLC50 family of uniporters (see Ref. 127). Transepithelial
glucose transport in the intestine and the kidney occurs by the
coordinated action of SGLTs through the luminal membrane
and GLUTs through the basolateral membrane. In the mam-
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0
Review
R1392
KIDNEY OF CARTILAGINOUS FISHES
Perspectives and Significance
The molecular anatomical approaches described in this review
contribute powerfully to the understanding of kidney function in
cartilaginous fishes. To propel this research strategy even further,
however, improvement of the “-omics” data is indispensable. In
cartilaginous fishes, only a limited number of databases are
available (elephant shark, http://esharkgenome.imcb.a-star.edu.
sg/; little skate, http://skatebase.org/). Comprehensive and largescale analysis of genomes and transcriptomes by nextgeneration sequencing will become a powerful tool to resolve
this problem. Research on the vascular system is also necessary. As described in Morphological Features of Cartilaginous
Fish Kidneys, contradictory observations have been reported
on the vascular system in the bundle zone (46, 71). The biggest
challenge to understanding the urea reabsorption process is
solving the mechanism for the “uphill” urea transport that is
required from the primary urine to the circulation where a high
concentration of urea already exists. Knowing the urea concentration gradient, researchers hypothesized the existence of
an energy-coupled urea transport system. Also the finding of a
unique central vessel (46) provided a new concept in the renal
vascular system and thus offered fresh insight into a urea
reabsorption model (see Fig. 6). After identification of transport proteins in each nephron segment, direct measurement of
the transport activities using purfused single tubules is also
required to test transport models. Collectively, integrative
analyses by anatomy, morphology, physiology, and molecular
biology of nephron and vascular systems will clarify the
physiological processes that take place in the highly elaborate
kidney of cartilaginous fishes. In addition, development (44,
111) and evolution of the nephron system are also of great
interest. These understandings will shed a new light on the
evolution and adaptation of this early diverged class of jawed
vertebrates, the cartilaginous fishes, over the course of earth
history.
ACKNOWLEDGMENTS
The authors thank Dr. Christopher A. Loretz of State University of New
York at Buffalo for critical comments on the manuscript.
Present address of K. Kakumura: Dept. of Aquatic Bioscience, Graduate
School of Agricultural and Life Sciences, University of Tokyo, Bunkyo, Tokyo
113-8657, Japan. Present address of Y. Yamaguchi: Hawaii Institute of Marine
Biology, University of Hawaii, Kaneohe, HI 96744.
W. Takagi is a Research Fellow of Japan Society for the Promotion of
Science.
This paper was presented, in part, in the symposium, “Adaptations for Salt
and Water Balance in Vertebrates” at the International Conference on Comparative Physiology and Biochemistry, held in Nagoya, Japan, May 31-June 5,
2011.
GRANTS
The works in this paper were supported by Grant-in-Aids for Scientific
Research and the Japan-Australia Research Cooperative Program from the
Japan Society for the Promotion of Science (JSPS) to S. Hyodo and by
Grant-in-Aid for JSPS fellows to W. Takagi.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: S.H. conception and design of research; S.H., K.K.,
W.T., K.H., and Y.Y. interpreted results of experiments; S.H. drafted manuscript; S.H., K.K., W.T., and Y.Y. edited and revised manuscript; S.H.
approved final version of manuscript; K.K., W.T., K.H., and Y.Y. performed
experiments; K.K., W.T., K.H., and Y.Y. analyzed data; K.K., W.T., K.H., and
Y.Y. prepared figures.
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