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The structure of the nephron in the south american lungfish lepidosiren paradoxa.

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Department of Anutomg, Harcard Medical School
This study of the kidney of Lepidosiren paradosa (South
American lungfish) had been largely completed when Bargmann’s ( ’34) excellent paper on the subject appeared. However, since our findings differ somewhat I’rorri his, and since we
have made more extensive observations upon some aspects
of the problem than he did, thc resnlts arc given liere in detail. Bargmanii gives a complete snrvey of earlier literature
on the kidney of the lungfish, and presciits fnlly his macroscopic findings, to which we have nothing to add. M7e vill
confine ourselves, therefore, t o a consideration of thp histology
of the nephron, with a comparative survey and interpretation of the resnlts of Bargmann and of Cordier (’29) upon
the iiepIiroris of Lepidosiren and Protopterus sp. (African
lungfish), respectively.
In a general study of the finer structure of the vertebrate
kidney, thc lungfishes are of particular interest in view of
their peculiar positioii in phylogeny. Fnrthermore, Smith
(’30) has brought out the striking fact that in the period of
estivation there is a complete cessation of urine elimination,
with the developmcnt of a marked uremia, a i d that the accumulated nitrogenous compounds are rapidly excreted soon
after the lungfish is returned t o water. Obviously, a knowledge of the detailed histology of tlic nephron is essential if
we are to arrive at any satisfactory correlation of morphology
and physiology.
OC‘lOlIBK, 1935
Through the courtesy of Dr. Homer W. Smith, of New York
University, and the New York Aquarium we were able to
obtain two sexually mature, non-estivating specimens of
Lepidosiren paradoxa, apparently in good condition and full>fed, one a male 20 inches long, the other a female, weight 350
gm. Fixatives used were Bouin’s, Zenker-formol and
Champy’s fluid. Serial sections were cut at 3, 5 and 10 u and
stained with If. and E., iron hematoxylin and orange G , and
IIeidenhain-azan. From 5 p sections of Rouin ’s material
stained with H. and E. the following wax-plate reconstructions were made, after the method of Born: one complete
ncphron (male specimen) at X 200, which was used as a
basis for study of the subdivisions of the nephron as seen
in all of the different preparatioiis; one nephron down t o and
including the transition from the first to the second portion
of the proximal; and two separate clusters of glomeruli, to
demonstrate the arrangement of the tufts and gross vascular
supply. The diniensions of the nephroii given below represent
an average of measurements taken from these models and of
many direct measurements from sections. From camera
lucida drawings of serial sections of Bouiii’s material stained
with azan (5 u ) , a graphic reconstrnction was made of the
entire vascular bed of one pair of glomeruli. Persistent attempts to isolate complete nephrons from material macerated
by the usual hydrochloric acid technic were unsiiwessful, due
in part to the fragility of the tissue and in part to the COMplication of the large amount of pigment presei1t.l
‘Within thc parenchyma of the kidneys of both of our specimens werc secn a
number of calcified parasitrs, in gencral oroid i n shape, varying i n avcrage
diameter from 5 t o 200 p . Thcsc werc located mainly in the supporting tissue,
but ofteii caused iiidcntatioi1s of the neighboring tubules and oceasioiially protruded through the tubular epithelium into the lumcn. Such tubules appeared
quitc uiidattiaged cxeept locally and 110 central or peripheral degenerative changes
were noted. The proliferative reartioii of connective tissue excited by the
parasisitcs was very slight, consisting of one very thin layer of fibroblasts siirrounding each calcified mass, with no giant cells nor any infiltration of inononuclear cells. No parasites were seen t h a t had not already uudergonc calcification,
and, except as noted, there was no morphologically appareiit damage doiie to
the kidiieys by their presence.
The nepliron of Lepidosiren paradoxa is divisible into the
following parts :
Average dimension8
in miUimetsr~
1. Glomerulus (iiidividusl tufts)
2. Neck segmciit
3. Proximal comoluted tubule
a. First portion
h. Second portion
4. Ciliated intermediate segment
6 . Distal convoluted tubule
6. Initial collecting tubule
Two views of a wax reconstruction of a complete iiephron
are given in figures 1 and 2. The tubule is connected with
onc of a pair of glomeruli, and the single afferent ( a ) and
efferent (e) vessels supplying botli tufts are represented. A
portion of the collecting duct, into which other tubules drain,
is included. Figure 3 is a schematic drawing, made without
regard to tlie relative diameter of the segments, in which the
convolutions of the reconstructed tubule are analyzed and the
course and extent of the differentiated segments are clearly
indicated. Fignre 4 is a simplified diagram of tlie average
nephron, drawn accurately to scale. Tn this figure it is to
be noted especially that the transition from the second portion of the proximal tubule to the ciliated intermediate segment is not abrupt with respect t o the outside diameter of the
tubule. Though an abrupt diminution in caliber might be
cxpectecl from the figures in the ahove tabulation, it must be
emphasized that these are only the average dimensions. As
noted below, the second portion of the proximal tubule is
smaller at the distal than at the proximal end. The usual
finding in a large number of tubules examined ~ 7 a sa gradual
diminution in diameter at the point of transition, and this
is indicated in figure 4. The numerical designations of the
segments in figures 3 and 4 correspond to those given in the
tabulation above.
G 1o wte YZI 1u s
The glomeruli occur regularly in clusters, which may inelude from tmo to twelve glomerular tufts (figs. 1, 2, 5, 6
and 8) ; only once x7as an isolated glomerulus noted in the
Figures 1 t o 3
entire study. Each glomerulus is roughly ovoid in shape, and
its size is independent of the number of tufts in the cluster.
Within each cluster tlie tufts are continuous (fig. 8 ) ; the
parietal epithelium of each glomerulus is fused with that of
the adjacent glomeruli at the point of reflection of the visceral
epithelium, but the intracapsular space associated with each
tuft is entirely separate from the others and drains into its
own tubule.
The nnmbcr of aff erait aiicl cff cirent vessels
with a 9roup of glomeruli varies
widely with dilfcrent ellisters ; in 110 case, hoJvever, does the number of either type of vessel
exceed the number of tufts lmseni. Tn a
number of cases when tlie vessels were traced
away from tlieir point 01 conncclion with the
tuft (i.e., llie afferent toward its origin from
a larger arterial trurik and thc efferent to its
point of subdivision iiito perituhular capil34
laries), the absence of snp connection betwren
them in the iiatiirc of an arterio-venous anastomosis was established. Wax reconstrucfioii of
a group of four glomeruli (fig. 5) demonstrated
only one afferent and efferent vessel, which
no. 1 in close proxienter and l e a ~ glomerulas
mity t o each other. All of the tufts, except 110s.
1and 4,are connected by braiicbes 01the VRPCUlar bed; hence, f o r blood to reach tuft no. 4 it
must first pass through two other tufts. In the
Coiirse of the study niimerous branchings of tlie
glomerular capillaries were noted. In the
rroup of six glomeruli illustrated in figure 6,
there are three afferent vessels, which arise as
tlie terminal branches of a larger arteriole at
4 a short &starice from the chstcr, and four efferl?nt s~essels.
The entire capillary bed of a pair of glomernli
Figure 4
mfis graphically rccoiistrncted from camera
lncida drawings (fig. '7) The diagram shows
the relative lengths of tlie capillaries only as projected onto
a plane siirface, and does not represent the true lengths. The
complexity of the vascular bed and the presence of numerous
capillary brancbings are apparent at once. The diagram
shows that the blood is supplied to both glomer~diby a common afferent vessel, and also removed by a common efferent
vessel. A striking finding is the existence of a very short
capillary connection (i.e., actually short in the original tufi)
betweeii the main afferent and efferent vessels soon after the
former enters the first glomerulus. This short loop is in the
nature of a potential shunt for the entering blood (see Bieter,
'30, for a description of ' shunt arrangements in the glomeruli
of the frog's kidney). Histologically the glomeruli have the
Fig.5 Drawing of a wax reconstruction of a cluster of four glomeruli. See
text. a, afferent; e, efforent vessel. Each glomerulus drains into a separate
tubule. x 100.
Fig.6 Drawing of a wax reconstruction of a cluster of six glomeruli. See
text and figure 8. x 100.
usual structnre ; they are markedly lobulated, show no thickening of the visceral epithelium, and are complctely vasculariaed, with no avascular central cellular core, such as is found
in the glomeruli of reptiles and birds (Corilier, '28 ; Marshall
and Smith, '30; Vilter, '35).
Neck segment
The transition from the parietal epithelium of the glomerulus to the ciliated epithelium of the neck segnent is sharply
defined. This segment (fig. 9) is slightly larger and the cells
are packed more closely at its proximal than at its distal end;
on the average there are seven or eight cells in a cross section
a t the proximal end, and four or five at the distal end. The
boundaries between the cells are not very distinct, but can
usually be seen. The nuclci are irregularly ovoid in shape,
with a fairly heavy chromatin network and usually contain
one or two heavily staining chromatin masses. The cptoplasm is not abundant, especially at the proximal end of the
segment, and is so very faintly granular as to appear almost
homogeneous. Cilia, fairly heavy and usnallp matted, arise
Fig. 7 Graphic reconstruction of the vascular bed of a pair of glomeruli.
See text.
from a small 'cap' a t thc top of each cell which stains more
darkly than the subjacent cytoplasm. I n favorable preparations each cilium can be seen to arise from a distinct basal
Pro%im a1 c O'IZ v o 1zct ed t ub ule
First porfioia. The transition from the neck se,gment to the
first portion of the proximal convoluted tubule is sharply
delineated, as regards both the type of epithelium and the size
of the tubule. This segment (fig. 10) is fairly regular in
outside diameter and the lumen is usually large, but undergoes considerable variation because of the variable height of
the cells. There are, on the average, six to ten cells in each
cross section of the tnhnle. The epithelium is sometimes low
cnhoidal, sometimes higher, and the sarfacc of each cell may
be either concave or convex. The cell boundaries are very
distinct. The details vary considerably in differrnt parts of
the segment, but the cssential striicture remains the same and
there is only one fundamental type of cell. The nuclei, usually central in position, are round and very regular, containing from six lo ten clistinct chromatin masses, with a moderately delicate chromatin network. In preparations stained
tvith iron hcmatoxyllin aftel. fixation in Champp’s fluid, the
iiuclei of this segment are undiffereiitiated when the nnclei
of all the other stlgments are brought out in dctail.
The cytoplasm stains rather l ~ e a ~ i land
p consists of a
gronnd network of finer grandes, and in addition many larger
granules o r inclusions, which arc much more numerous in the
luminal portion of the cells and are of variable size. M7ith
thc azan stain the ground cytoplasm stains orange-red and
the larger inclusions blue. Vacuoles are freqneutly observed.
A number of round, yellow inclusion bodies of varying sizes
arc found in this segment, usually lyiiip in the basal part of
the cell, and occurring in certain sections of the segment and
not in others. A brash inorder appears essentially throughout, lint with great variation as t o both lieiglit and density,
I n some places it is exceptionally tall and lleautiful, in othcrs
its existence is doubtful. At times it is so densc as to appcar
almost homogeneous, especially where it is hiyb : at other.
times it is low and the single elements of the ‘brash’ stand out
very distinctly. When the cell has a convex Farface and protrudes into the lamcn, brush may be present on both the top
aad the sides of the ccll. in which case it is often higher on
the top than on the sides, or it may be absent from the sides
altogether. From some cells delicate, slender cilia-like processes can be seen to project beyond the lnrush border into thc
lumen. The processes suggest, but are in no way so characteristic or distinct as, the true cilia found clsewlicre in the
nephron, and the true naturc of this modification of the cell
bordcr is not clear.
KO subclirision of tbis portion of the proximal convoluted
tubule can he made. The variations appear t o be haphazard
and t o merge gradually into onc another. S o correlation could
be found between the height of the cells, the height of the
brnsh border, the nature of the granules, the presence of
cilia-like processes or the presence of the y c l l o ~inclusion
Secofad y o r t i o i ~ The transition between the first and
second portions of the proximal convohted tubule is sharp.
This secorid portion (fig. 11) is fairly uniform in outsidc
diarneter, being somewhat smaller at its distal than at its
proximal eiid. The himen is miich smaller than in tlie first
portion, averaging 25 p, but in places being its narrow as 5 p.
All intercellular boundaries are quite distinct. The epithelium is composed of tliree cell types, the ‘light,’ ‘brush’ and
ciliated cells. The basic: cell is the ‘lighl’ cell, rhich occiirs
predomiiiantly thronghout the entire segment. These cells
are irregular in shape and in places protrudc uiieveiily into
the lumen. As the lumen is relatively small, the base of the
cell is niach broader than the apex in cross sections of the
tubule. The abundant q-toplasm, suggesting a network of
fine interlacing lines, is denser toward the lumen than at the
base, and in all preparations stailis quite lightly. The lnminal
border of the cells stains comparativelv darkly, and in some
places exhibits an appearance with vertical striations which
is to be iiiterprebed as a very IOW brush border. The nuclei
are usually located near the base, but the position is quite
variable. They are much like those of the first portion, containing six t o ten chromatin masses, but the chromatin network is not so dense. Typical isolated ‘brnsh’ cells, morphologically identical with those of the first portion, occur only
within the proximal half millimeter of this segment. They are
relatively few in number, but in some places there are several
to a single cross section. KO cells of this type were ever
found distal t o the first half millimeter. Throughout this
second portion of the proximal convoluted tubule there occur
isolated ciliated cells which increase in frequency along the
segment, averaging one cell to three 5 p cross sections at the
proximal, and about three cells to one cross section at the
distal end. They are roughly triangular or trapezoidal in
shape, almost always occur between ‘light’ cells, and only
about half of them extend down to the basement membrane.
The nuclei, irregularly ovoid in shape, are located near the
luminal border, and present a much darker, granular. chromatin network than the ‘light’ cells, with one to three chromatin
masses. The finely granular cytoplasm is rather scanty
and stains darkly in all preparations. Each cell has a bundle
o f cilia arising from a darkly staining ‘cap’ on its lnminal
surface, similar to the ciliated cells in the neck segment.
Ciliated ivtterrnediate segment
The transition from the second portion of the proximal to
the ciliated intermediate segment is quite sharp. The cells
of this segment (fig. 12) are low cuboidal and very small, so
that, although the outside tubular diameter is markedly
diminished in comparison with the preceeding segment, the
lumen diameter is comparatively large, averaging about 20 p.
The luminal border of the cells, which are poorly delimited
from each other, is very flat, giving a smooth, regular lumen.
The scanty, faintly granular cytoplasm stains almost homogeneously. The nuclei are round or ovoid, slightly irregular,
and relatively large, covering almost the entire cell. Each
contains three or four small chromatin masses and a light
granular chromatin network. A very sharp differentiation
of this segment is obtained in iron hematoxylin preparations
of Champy material, when the nuclei, with their light brown
background, stand out in marked contrast t o those of other
segments, and the cilia are very distinct. Cilia arise from
the center of the luminal border of each cell, fuse into a small
clump, and extend down the lumen f o r a distance of about
one cell and a half. The appearance is very regular and
characteristic, as is well shown in figure 12. The cilia do not
extend into the next segment more than the length of one or
two cells.
L) ist a1
cowu o lzct e d t ubzcl e
The transition from the ciliated intermediate to the distal
segment is sharply- dcfincd. The cells of this segment (fig. 13)
are cuboidal, of medium height, and the lumen, which is of
moderate size, is usually quite round, although in places the
cells protrude into it in gentle curves. There arc somc variations in the size of this segment, but they are small, and the
general appearance is remarkably regnlar and constant. The
deiise cytoplasm stains deeply and gives a striated appearance, the striations being arranged perpendicular to the base
of the cell. I n places there are unstained vacuoles of varying
sizes within the cytoplasm, usually in close proximity to the
basal border of the nuclei. Also, yellow inclusion bodies,
similar to those noted in the first portion of the proximal, are
sometimes seen in this segment. Cell boundaries are fairly
distinct. The nuclei, round, fairly large and centrally placed,
show fit7e or six chromatin masses, often with a tendency t o
be arranged peripherally. Radiating from each main chromatin mass are delicate chromatin fibrils, and covering the whole
nucleus is a faint, irregular chromatin network.
I d i a l collecti9t.q tubule
The transition from the distal se,gnent to the initial collecting tubule is quite gradual, so that a more or less arbitrary
division has to be made in order t o obtain their respective
lengths. The nuclei are on the average more numerous in
cross section, the cells are aomewhat more compressed, and
the cytoplasm is correspondingly less in amount (fig. 14).
Cell boundaries are not so distinct as in the distal, and by
comparison this segment is rather irregular in appearance.
The cytoplasm is almost homogeneous, showing very faint
striations. The nuclei, round, ovoid o r wedge-shaped, are
centrally placed and show a variable number of chromatin
masses (about six 011 the average) which again often tend to
be arranged peripherally.
Our results are similar in most essentials to the findings of
Cordier (’29) in Protopterus and Rargmaiin (’34) in
Lepidosircn paracloua. However, there are some significant
differences which must be noted. We found the number of
glomeruli in a single cluster to be as high as twelve, whereas
13argmann gives tlic range to be three t o fire, Cordier three
t o four. It must be emphasized that the number of tufts in
8 section of a given cluster at its greatest diameter may give
a totally erroneous idea of the total number present. The
arerage glomerular dimeiisioiis in OUT material (180 x 200 u)
are considerably greater tlian those giveii h y Eargmann (100
X 140 p) and Cordier (140 p ) . Alarshall and Smith ( ’30) gi17e
153 p as the average size in T’rotopterus aethiopicns. With
Bargmann we found tlic number of glomeruli to he relative1:large, while Cortlier reports them t o be few in number in
Protopterus. Rargmaiin speaks of a vascular pole of the
glomeruli and suggests that the glomeriilar bed consists of a
single unbranched convoluted capillary. We liave found the
p o s s vascular supply of the clusters to be most irregular, and
the presence of numeroils branchings of the glomerular capillaries has been adequately demonstrated. The peculiar and
striking characteristics of the glorneunli in this species must
be borne in mind in any attempted cvaluation of glomerular
function both under normal condi tioris and in estivation.
O w first portion of the proximal convoluted tubule corresponds to the ‘tubule contoum6’ of Cordier a n 4 the ‘Hanptstiick’ of Bargmann. No diverticula of this segment, such as
are reported as a rather constant feature in Protopterus by
Cordier, were seen in our material, but no extended search f o r
them was made. Although we did find numcrous small inclusions in the supranuclear zone and marked variation in the
type of brush border, me did not find any very large ii~clusions
nor any cells which could be said to have absolutely no trace
of brash border, both of which are beautifully pictured by
Our second portion of tlie proximal tubule corresponds to
the ‘segment rectiligne’ of Cordier in Protopterus, with these
differences in the two animals, that Cordier found none of o u r
‘brush’ cells and describes oiily one fundamental type of cell
in the entire segment, with a low and often almost indistinguishable brush border, and with cilia arising from certain
of these cells i n the distal end. Since he could find no ‘segment reetiligne’ in young, immature females but did find it in
his mature males, he cautiously suggests the possibility that
it might be a sexual segment. Siiice this segment is identical
in our male and female specimens, we conclude that it is not
a sexual segment in Lepidosiren. Our second portion of the
proximal also corresponds to the ‘sog. uberleitungsstuck’ of
Bargmarin, arid his description is in accordance with our observations with the exception that he found none of oily
‘brush’ cells. The cliaracteristics of this segment, when taken
in conjunction with our demonstration of a true ciliated intermediate segment (Corclicr’s ‘segment grcle’ in Protopterus),
justify the conclusion that it unquestionably represents a
markedly modified portion of tlie proximal convoluted tubule.
Rargmann came t o the sariie coriclnsion, but denies the presence of a ciliated iiitermediate wgnient in his material.
We did not encounter any of the diverticula reported by
Cordier f o r the distal segment in Protoptcrns. It is t o be
noted that this author suggests, on the basis of its cytological
eliaracteristics, a resorptive fiinction for t i e initial collecting
tubule (segment d ’union).
I n conclusion, I wish t o thank Dr. Allan L. Grafflin for suggesting this problem, and for his continned advice aiid
criticism t,hronghout the investigation.
The nephron of Lepidosiren paradoxa is divisible into the
following parts : glomerulus, neck, proximal convoluted
tubule, ciliated intermediate segment, distal convoluted tubule
and initial collecting tubule. The glomeruli occur typically
in clusters, with a variable and irregular blood supply and a
branching capillary bed. The proximal convoluted tubule is
divided into two portions. The cells of the first portion are
provided with a typical brush border, and arc comparable
with those of the proximal convolution as seen in other vertebrate kidneys. The second portion has as the fundamental
cell type a large ‘light’ cell, which sometimes shows a low
brush border ; in addition, isolated ‘brush’ cells, identical with
those of the first portion, are present in the first half millimeter of the second portion, and isolated ciliated cells occur
throughout its length. The demonstration of a true ciliatcd
intermediate segment establishes this second portion as a
markcdly modified segment of the proximal convoluted tubule.
W. 1934 Untersuchungen iiber Histologie unil Histophysiologie
der Fischniere. 1. Dipnoer: Lepidosiren paradoxa. Zeit. f . Zellforsch. 11. mikr. Anat., vol. 21, p. 388.
R. S . 1930 The effect of the splanchnics upon glomerular blood flow
in the frog’s kidncy. Am. J. Physiol., vol. 91, p. 436.
Corln~nt, R. 1928 $;tudes histophysiologiques sur le tube urinairc des reptiles.
Arch. de Biol., vol. 37, p. 1.
1929 Le tube urinaire d u ProtoptBre (Dipneuste). Compt. rend.
de 1’Assoc. des Anat., 24‘ RBunion, Bordeaux, p. 157.
E. K., JR.,AND H. W. S h i r m 1930 The glomerular development of
the vertebrate kidney in relation to habitat. Biol. Bull., vol. 59, p. 135.
S m w , H. W. 1930 Metabolism of the lungfish, Protopterus aethiopicus. J.
Riol. Chem., vol. 88, p. 97.
VILTER, R. W. 1935 The morphology and development of the ~nctauephric
glomerulus in the pigeon. Anat. Rcc. (111 press.)
8 Section through the cluster of six glomeruli picturcd i n figure 6. Note t h a t
the tufts, four of which appear in the section, are united centrally, while the
iiitracapsular spaces are separate and distinct. Bouiii’s, $1. slid E., 5 ~ .X 200.
9 Longitudinal section of tlie ciliated iicek segment. Champy, iron hematoxylin, 5 p . x 800.
10 Cross section of tlie first portion of the proximal convoluted tubule. The
briish border is clearly .risible. Bouiii’s, iron hematoxylin, 5 p . X 480.
11 Cross section of the secoiid portion of the proximal coiivoluted tubule, showi n g the ‘light’ cells w t h their abundant, pale cgloplasni and basal nuelci. Part
of’ ail isolatcd ciliated cell is seen to the right with its nucleus 1)iiig c.lose t o the
lumen. Thc cilia do not appear in this section. Bouin’s, 11. and E., .5 p. X 480.
12 Longitudinal section of the ciliated intrrrnediate segment. Champy, iron
hematoxylin, 5 p . X 800.
1 3 Cross section of the distal convoluted tubule, showing the dense cytoplasm
and the characteristic verticd striations. (Thampy, iron llenistox~lin,5 p. X 480.
1 4 Longitudiiial sectiou of the initial collwting tubule. Cliainyy, iron hematoxg-lin, 5 p. X 480.
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lepidosiren, structure, nephron, lungfish, south, paradox, american
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