Zare-41
3/March
2011/5/-41
Journal
of the
Persian Gulf
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(Marine Science)/Vol. 2/No. 6/December 2011/7/45-52
Ultra Structure of Excretory Organ (Kidney)
in Juvenile Grouper, Epinephelus coioides
Rumiani, Ehsan1; Abdi, Rahim1*; Zolgharnein, Hossein1;
Savari, Ahmad1; Morovvati, Hasan 2
1- Department of Marine Biology, Faculty of Marine Science, Khorramshahr University of Marine Science
and Technology, Khorramshahr, IR Iran
2- Department of Histology, Faculty of Veterinary Medicine, Ahvaz University of Shahid Chamran,
Ahvaz, IR Iran
Received: May 2011
Accepted: October 2011
© 2011 Journal of the Persian Gulf. All rights reserved.
Abstract
In order to study of kidney tubules, twenty specimen of 45 days old juvenile grouper
(Epinephelus coioides) were prepared after proper biometry. Five mm thick sections were
removed from the middle and posterior end of the kidney and fixed in Bouin and
glutaraldehyde. The routine procedures of preparation of tissues were followed for light and
transmission electron microscopic study. In macroscopic observations, it was observed that
kidney was located retroperitoneal and attached to the vertebral column with an average length
of 4±0.2 Cm. The microscopic results showed that nephrons consisted of glomerules, proximal,
distal and collective tubules. The proximal tubules were detectable with their brush border.
Distal tubules had cuboidal cells with round nucleus that were positioned in the middle part of
the cell. The lumen of these tubules were PAS negative due to lack of brush border. Collecting
tubules were also PAS negative as their cuboidal cells lacked brush border and their ellipsoid to
round nuclei were positioned central to the cell. TEM analyses of sections showed more details
of tubules structure, e.g. plasma membrane showed an extensive system of depressions in all
tubules, except in collecting tubules.
Keywords: Ultra structure, Excretory organ, Grouper, Epinephelus coioides.
1. Introduction
Grouper fishes belong to Serranidae and live in
waters of countries south-east of Asia. Groupers are
environmentally hypo-osmotic marine fishes. These
fish species lose water and uptake salt actively. Instead,
fishes drink water and actively excrete monovalent ions
*
E-mail: [email protected]
through the gills and some bivalent ions through their
kidney (Cataldi et al., 1995; Grindstaff et al., 1996;
Heemstra and Randall., 1993). Kidney is very
important for osmoregulation in marine fishes, and
does this by changes in rate of urine secretion and
balance between secretion and reabsorption (Hickman,
1968; Lin et al., 2004). Nephron in bony fishes is
composed of glomeruli, a ciliated neck segment, a
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Rumiani et al. /Ultra Structure of Excretory Organ (Kidney) in Juvenile Grouper, Epinephelus coioides
capsule, endothelial cell, mesangial cell, visceral and
parietal epithelium of the renal capsule, podocyte cells,
vascular bridge and capillaries (Fig. 1).
proximal tubule, an intermediate segment, a distal
tubule and a collecting tubule. Kidney in teleost is
without henle arcuate and some are without glomeruli
(Hickman et al., 1969; Hoar et al., 1983). In fish,
maintenance of blood osmolality and ion concentration
is performed differently than to the external
environment (Nebel et al., 2005). This is based on
active transport of ions particularly Na+ and Cl- by
absorption and excretion depending on the salinity of
environment (Lin et al., 2004). Because of the
importance of these fishes in nutrition and
osmoregulation, this study was performed.
The neck segment connecting glomeruli to first
proximal tubule, composed of short cuboidal
epithelial cells with round nuclei located basally
(Fig. 2).
2. Materials and Methods
Fish samples were colleced from Imam Khomeini
Marine Research Station, affiliated with the Institute of
Aquaculture in South of Iran. For this purpose, 20
juvenile grouper with 5±0.1 g weight were prepared
and then anesthetized by carnation.
Then, middle and posterior part of kidney were
removed, fixed in the Bouins solution and transferred to
the histological laboratory. After 24 h, samples were
removed from Bouin solution cleaned up and washed
in 70% ethanol multiple times. The routine procedures
of preparation of tissues were followed and paraffin
blocks were cut in 6 micron sections, stained with
H&E, PAS and then, studied under light microscope
(Hickman and Trump, 1969).
Some samples were prepared for TEM analysis.
These samples were fixed in 2.5% glutaraldehyde
primary fixative for 8 h and in secondary fixative for
12- 24 h (Sodium Phosphate 1%). Finally, sections
were prepared routinely for TEM analysis (Caberoy
and Quinitio, 2000).
Fig. 1: Cross section glomeruli of kidney in (E. coioides). Bouin,
H&E, ×100. 1. Podocyte cell, 2. Visceral epithelium of renal
corpuscle, 3. Parietal epithelium of renal corpuscle, 4. Blood
capillaries, 5. Endothelial cells, 6. Mesangial cells, 7. Urinary space,
8. Vascular bridge.
Fig. 2: Cross section of mesonephric part of kidney in (E. coioides).
Bouin, H&E, ×40. NS: neck segment, PI: first proximal tubule, PII:
second proximal tubule, C: collecting
3. Results
Proximal segments I and II, consisted of cuboidal
cells basally positioned nuclei but in PII was drown
toward to the middle part. Also, collecting tubules
were PAS negative as their cuboidal cells lacked
brush border and their ellipsoid to round nuclei were
positioned central to the cell (Fig. 3).
Results of microscopic study indicated that nephrons
in grouper kidnies consisted of glomerulus, neck
segment, proximal, distal and collecting tubules.
Glomeruli included Bowman's space, Bowman's
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In PAS staining, proximal tubules were positive
by their microvilli (Fig.6).
Fig. 3: Cross section from mesonephric part of kidney in (E.
coioides). (P): proximal, (D): distal and (C) collecting tubules.
Bouin, H&E, ×40.
The cells were longer in PII than the PI. There was
brush border in both segments on apical surface of
epithelia, extended into the lumen but in a reduced rate
from segment I towards the segment II (Fig. 4&5).
Fig. 6. PAS staining, cross section of kidney tubules in (E.
coioides) showing both proximal segments (PI & PII) with their
brush border (BB) and distal tubule (D). Bouin H&E, ×40.
Distal tubules had cuboidal cells with a round
nucleus positioned centrally to the cells and tended
to be larger in cross section. Lumen of tubules were
PAS negative due to lack of brush border . As
mentioned, collecting tubules with their cuboidal
cells, lacked brush border, were PAS negative and
their ellipsoid to round nuclei positioned central to
the cell (Fig. 7).
Fig. 4: Cross section of first Proximal tubule. (PI). Bouin, H&E, ×40.
Fig. 7: PAS staining .cross section from collecting tubule (C).
Bouin H&E ×40.
The height of cells in collecting tubules were greater
than distal and proximal tubules but conversely, the
diameter of lumen of the former was smaller.
Fig. 5: Cross section of second Proximal tubule. (PII). Bouin,
H&E, ×40.
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In Transmission Electron Microscopic study, five
different segments were distinguished: (1) neck
segment (2) first proximal segment, (3) second
proximal segment, (4) distal tubule and (5) collecting
tubule, which joined to a collecting duct.
The wall of cells in proximal tubules were
extensively invaginated along their basolateral
surfaces. Mitochondria were distributed in cytoplasm
except in the area below the brush border (Fig. 10).
Neck segment: The epithelial cells of this segment
were short-cuboidal with round nuclei and a few
number of mitochondria. Cell membrane depression
in the basal or lateral portion was shallow and the
apical surface of cells showed no brush border but
short microvilli and cillia were visible (Fig. 8).
Fig. 10: Electron micrograph of tubular epithelium. IV:
invaginations, M: mitochondria, BM: basement membrane, SM:
smooth muscle. Magnification: ×9800.
Second proximal tubule: The epithelium of this
segment like the first proximal segment consisted of
cuboidal cells that were longer and with low- density
brush border. Also, the nuclei of the cells were
oriented toward the center. Other specifications of
the cells were similar to that of the first proximal
tubule (Fig. 11).
Fig. 8: Electron micrograph of renal tubular epithelium (neck
segment). TL: tubule lumen, N: nucleus, M: mitochondria, C: cilia,
m: microvilli, IF: infoldings of membrane. Magnification: ×2750.
First proximal tubule: The cuboidal cells of this
portion had round nuclei with a well-developed
brush border in the apical surface. There were
numerous tightly packed microvilli more than the
other segment (Fig. 9).
Fig. 11: Electron micrograph of second proximal tubules. TL: tubule
lumen, BB: brush border, C: cilia, N: nucleus, M: mitochondria, L:
lysosome, IV: invaginations, BM: basement membrane, SM: smooth
muscle. Magnification: ×7000.
Fig. 9: Electron micrograph of first proximal and distal tubules. TL:
tubule lumen, N: nucleus, M: mitochondria, BB: brush border, L:
lysosome, BM: basement membrane, SM: smooth muscle.
Magnification: ×2750.
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Journal of the Persian Gulf (Marine Science)/Vol. 2/No. 6/December 2011/7/45-52
Distal tubule: As seen in TEM pictures, in this
rigion, cells were cuboidal and larger than other
parts. Mitochondria were distributed into the
cytoplasm. Epithelial cells were cuboidal with
elliptical to round nuclei centrally in the cell. Also,
this segment lacked brush border and basolateral
invagination of membrane was more observable
compared to other segments.
Collecting tubule: Collecting tubules recognized by
high-cuboidal cells with round nuclei positioned
centrally with no brush border. Also, basal membrane
invaginations were observed in other tubules (Fig. 12).
morphological characteristics. Like the osteichthyans
and teleostean, the kidney of E. coioiodes is divided to
three sections: the head, trunk (mesonephric portion)
and caudal section. Histological studies of trunk in
juvenile grouper showed that nephrons consisted of
glomeruli, neck segment, proximal, distal and
collecting tubules. Renal corpuscle consisted of
Bowman's space and capsule, endothelial cell,
mesangial cells, parietal and visceral epithelium,
podocyte cells, vascular bridge and capillaries. The
glomeruli was a coil of capillary which occupied the
Bowman’s spaces (Hyman, 1942; Kumar and
Tembhre., 1996). The mesangial cells are barrier for
free molecules and ions during the filtration process
(Kumar and Tembhre., 1996). As, Hickman in 1969
described, mesangial cells are essential for rapid
adaptation to different environments. The presence of
mesangial cells in glomerulus in other species such as
Huso huso and Acipenser persicus was reported
(Hickman and Trump., 1969). Viviane and Carolina
(2006) described that the epithelial cells of kidney
(composed of mesonephric convoluted tubules) were
mitochondria-rich with plasma membrane depression
(Viviane et al., 2006). Basolateral membrane
invaginations were observed in all segments but with a
greater abundance in basal section. Neck segments with
short- cuboidal cells, were short and connected the
glomeruli to the first proximal segment (PI). Also
mitochondria were less frequent and positioned in apice
of the nucleus. Height of epithelial cells increased
gradually towards the first proximal segment.
Depression of membrane in basal and lateral portion
was shallower and the apical surface of the cells
showed no brush border but some short microvilli and
many cilia were present. These results coroborated
findings of Ottosen (1978) on marine flounder,
Pleuronectes platessa. As Kumar and Tembhre (1996)
reported Huso huso and Acipenser persicus proximal
tubules constituted cuboidal cells with basal nuclei with
a well developed brush border (Kumar and Tembhre,
1996). Due to their brush border, this tubules
differentiated from distal and collecting tubules by PAS
Fig. 12: Electron micrograph of collecting tubules. TL: tubule
lumen, N: nucleus, M: mitochondria, IV: invaginations.
Magnification: ×4900.
Features Commom to all Segments: All tubular
segments were surrounded by smooth muscle cells.
Mitochondria and basal membrane depression were
observed in all epithelial cells of tubules.
4.Discussion
About 5% of teleosts are euryhaline which can
tolerate salinity of their environment. Grouper is
euryhaline and lives in tropical and sub tropical region
(Ogawa, 1961). Adults live in rigid and coral reef area
while larvae and juvenile live on surface of aquatic
plants and estuarine (Caberoy and Quinitio, 2000). The
morphologic structure of kidney in marine organisms
was devided to five groups by Ogawa in 1961. Based
on this categorization, E. coioides has type III of
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Rumiani et al. /Ultra Structure of Excretory Organ (Kidney) in Juvenile Grouper, Epinephelus coioides
(Flik et al., 2002). This segment receives the filtrate
from the distal tubule and excretes it. Collecting
tubules were larger than proximal and distal tubules.
Large collecting ducts were formed by the fusion of
number of collecting tubules and drainage several
nephrons. Cells joined at the top with an occluding
junction. Occluding junctions are impermeable to
ions, and their presence implies that there's a good
reason to prevent free percolation of materials from
one side to the other (Youson et al., 1989). In
conclusion, the study clearly showed that ultra
structure of excretory organ in juvenile grouper was
identical to the previously description, presented for
different fish species.
staining (Tang et al., 2010). The proximal tubules of
euryhaline teleost have two segments, the first and
second (Hoar and Randall, 1983). Proximal tubule of
(E. coioides) as an euryhaline species, included two
segments like Elasmobranches, (Huso huso) and
(Acipenser persicus) (Tang et al., 2010). The number of
microvilli reduced from segment I to the segment II
(Charmi et al., 2010). The brush border of proximal
tubules was specializated for increasing surface area
for absorption. The difference between the first and
second proximal segments was not very pronounced,
but reduced in density of brush border and cells
nuclei toward the central section of the cell.
Ottosen (1978) showed kidney of Pleuronectes
platessa lacked distal tubules. But, the results of this
study on E. coicoides was similar to the findings of
Tang et al.(2010), Lin et al. (2004) and Charmi et al.
(2010). Other studies on Chanos chanos, Tetraodon
nigroviridis, Huso huso and Acipenser persicus
showed that distal tubules were present in nephrons
(Cataldi et al., 1995). Also, Hickman and Trump
(1969) reported that in the euryhaline environment,
southern flounder had both distal and collecting
tubules, but in this fish no intermediate segment had
been reported. Based on results of this study, like
Elasmobranches distal tubules in E. coioides had
cuboidal epithelium with no brush border (Viviane et
al., 2006). Round nuclei were specific of distal
tubules located centrally.
Distal tubules were not specialized for absorption
but functioned in pumping ions, for example sodium
out of lumen into the surrounding intracellular space,
via active transport pumps. In distal tubules, ion
passage control is vital, and the only way that ions
are allowed to excrete out of the tubule via controlled
active transport. The structure of collecting tubules
in E. coioides, because of longer cuboidal epithelium
with central nuclei differ from other tubules, similar
to sturgeons (Prodocimo and Freire., 2006; Olsen,
1970). In ultrastructural study on Pleuronectes
platessa a distinct segment with strongly basophilic
cells without any brush border had been described
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