Factors responsible for the abnormal distention of glomerular capsules in the Necturus kidney.

FACTORS RESPONSIBLE FOR THE ABNORMAL
DISTENTION O F GLOIIERULAR CAPSULES
I N THE NECTURUS KIDNEY
ALFRED M. LUCAS' AND H. L. WHITE
Department of Anatomy and Department of Physiology, Washington University
School of Medicine, St. Louis
THREE TEXT FIGURES AND ONB PLATE
The accessibility of glomerular capsules and different
regions of the renal tubule has led many investigators to
perform physiological experiments upon the amphibian
kidney. Failure to correlate certain morphological conditions peculiar to the kidneys of lower vertebrates renders
invalid in some cases the interpretation of the experimental
results.
It has been noted by White ('28) that the glomerular
capsule in Necturus may be distended with fluid, although the
formation of fluid indicated by the rate of glomerular circulation is depressed. When this condition occurs, colored solutions added to the ventral surface of the kidney pass from
the nephrostome upward into the capsule against the action
of the cilia which line the neck, as well as downward into the
proximal convoluted tubule. Not only may distended capsules be found in the primary tubules (those possessing a
nephrostome), but also in those which lack a nephrostome.
Consideration is given in the present investigation to each
of the various structural factors which might be responsible
for the abnormal accumulation of fluid in the capsule, namely,
the cilia, a blockage of the ureter, or a blockage of the renal
tubule.
'I an1 indebted to the Chemical Foundation and to the National Research
Council f o r financial aid in this investigation.
371
372
ALFRED M. LUCAS A N D H. L. WHITE
MATERIAL AND METHODS
The animals (Necturus maculosus Rafinesque) were obtained from Minnesota during the winter and early spring.
They were kept for from one t o three weeks in large tubs
filled with tap water, sometimes in the laboratory and sometimes in an unheated basement room, and in most cases
remained in apparently good condition, although we have
iiover been able to get them to eat.
They were anesthetized by immersion in 1.5 per cent
urethane solution, transferred to a board on which the upper
half of the body was immersed in water, and opened. The
ventral surface of the kidney was inspected by reflected light.
The various parts of the tubule were identified and the direction of fluid passage was determined by adding solutions of
trypan blue or nigrosin in frog Ringer’s to the ventral
surface of the exposed kidney.
Ciliary activity within the nephrostome and neck was ohserved on thin sections of living tissue with an 8-mm. objective
and transmitted light. The section was cut from the ventral
surface of the kidney by employing a sharp-edged revolving
disc attached to the handle of a dental engine and rotated at
a medium rate of speed. A cover glass was held against the
ventral surface of the kidney while the cut was being made,
and the free edges, not included in the section, were held by
forceps. The slow addition of Ringer’s to the moving disc
minimized the mutilation of the tissue. The disc, made from
sheet silver 0.004 inch thick, has a diameter of l % ~ inch.
Rigidity of the cutting edge is lost when discs of larger
diameter are used.
Fixed preparations were made by clearing the blood from
the vessels with frog Ringer’s followed by injection of Allen’s
B, modification of Bouin’s fluid (McClung, ’29). Sections
cut 5 CI thick were mounted serially. The structure of the
ciliary apparatus is best shown after Heidenhain’s iron
hematoxylin and acid fuchsin.
DISTENTION O F GLOMERULAR CAPSULES
373
OBSERVATIONS
Ciliary activity im the neck of the rerzal tubule
The glomerulus, capsule, ciliated neck, and proximal convoluted tubule of those uriniferous tubules which posses a nephrostome are easily visible in vivo. Normally, the diameter of
the capsule is usually not more than 50 per cent greater than
that of the glomerulus even with rapid glomerular circulation.
Dye placed on the ventral surface of the kidney passes
through the nephrostome into that portion of the ciliated
neck distal to their junction and thence into the proximal
convoluted tubule, only one or two seconds being required
for passage.
Dye entering the nephrostome of an abnormally distended
capsule is carried up to the capsule as well as away from it.
This takes place either when the capsule wall is intact or
after it is torn open. I n the latter event, a small droplet of
dye placed on the mouth of the nephrostome is carried up
and out through the rent. The upward flow of fluid into either
a torn or an intact capsule ceases if a hole is made in the
proximal tubule, but may be again induced by compressing the
tubule above the opening. Capsular distention may be present
when the animal is first opened, and frequently all of the
visible capsules are thus affected, or it may develop an hour
or more later, in which case the condition arises gradually
and usually in relatively few capsules.
Since reversal of ciliary movement has been described
(Parker, '05), it was necessary to determine whether this,
occurring in the proximal portion of the neck, caused the
retrograde passage of fluid into the capsule. Thin sections
cut parallel to the ventral surface of the kidney, removed
and placed in Ringer's solution on a slide, reveal under the
microscope the movements of the cilia on individual cells.
Such a longitudinal section of the ciliated neck is represented
in figure A. Normally, the basal third of the compound cilium
extends toward the center of the lumen and inclines distally
(figs. A and C and figs. 16,17, 19, 31, and 32 by Chase, '23) ;
the separated fibrils of the outer two-thirds lie parallel to the
374
ALFRED M. L U C A S AKD H. L. WHITE
axis (fig. A ) . The direction and restricted amplitude of
the active beat by the cirrus-like portion of the compound
cilium is indicated by arrow a in figure A. During both
active and recovery strokes an equally rigid form is maintained. The separated fibrils of the distal two-thirds have a
characteristic flagellar movement (fig. A, b ) which viewed
in optical section has the appearance of rapidly but more or
less regularly moving waves.
Fig. A Diagram of a ciliated neck viewcd longitudinally. The cilia from only
one cell are shown in their entirety. a, the amplitude and direction of the
effective beat belonging to the cirrus-like b a d third of the compound cilium;
b, the amplitude of the flagellar movements exhibited by the separated fibrils
of the outer two-thirds of the cornpound cilium.
The ciliated neek in the living section is usually collapsed,
in which case the cilia exhibit no perceptible activity. They
are always directed distally and compressed toward the walls.
The reaction of the neck and its cilia to fluid pressure is
revealed in the following manipulations. Dye added to the
surface and conveyed to the neck by the nephrostome moved
slowly up the neck toward the capsule, attended by dilatation
of the neck wall. Addition of Ringer’s failed to replace the
dye in the neck, but followed upward behind it. The region
last t o open and seemingly offering the greatest resistance
was the junction of the neck with the capsule, and only after
its relaxation did the capsule begin to fill. At the instant
DISTENTION O F GLOMERULAR CAPSULES
375
the neck lumen enlarged, the cilia in that area became fully
active ; the rigid basal portion maintained itself distally projected against the upward flow, but the ends of the cilia
seemed forcefully carried backward by the stream, which
resulted in curved and circular vibrations giving the optical
effect of a milling movement. When Ringer’s was no longer
added, the capsule collapsed in the succeeding fifteen to
twenty minutes. At the end of this period, the neck walls
contracted suddenly throughout their length and simultaneously the cilia ceased activity; even slight flickering movements at the tips of the cilia were inhibited.
Fig. B Ciliated cell from the neck region of a renal tubule. The plane of
section is the same as in figure A. Only the basal third of the compound cilium
is included.
The addition of Ringer’s, resulting again in the progressive opening of the neck and distention of the capsule, was
followed by cutting across the proximal convoluted tubule
with scissors; immediately the capsule emptied and the neck
collapsed. A needle pressed across the tubule above the cut
caused rapid dilatation of the neck and capsule. These
observations have been repeated on several animals.
The attachments of the cilia to the cell are shown in figures
B and C. A similar intracellular ciliary mechanism is present
in cells of all the ciliated segments of the uriniferous tubule.
376
ALFRED M. LUCAS AND H. L. WHITE
A densely staining ground substance surrounds the fibrils
and both terminate at a layer of intracellular granules. The
fibrils spread asymmetrically (fig. B) in a direction opposite
to that of the effective beat of the cilia. The intracellular
ciliary area and its contained fibrils in a transverse plane,
shown in the upper and right-hand parts of figure C, has the
form of a crescent and the fibrils are symmetrically arranged.
The granule attached to the basal end of each intracellular
Fig.C Cross section of a nephrostome near the mouth. The intracellular
apparatus is irregular and appears to extend into the nueleus in some regions,
because the plane of section passes obliquely across part of the wall.
fibril, together with the granule at the free surface of the
cell, may represent the two parts of a diplosome basal body,
which in these cells are widely separated. If such be the case,
the intracellular fibril is here not homologous with a true
ciliary rootlet. The suggestion is strengthened by the fact
that a true ciliary rootlet has not been described as terminating in a granule. The ciliated cells present in the kidneys
of ophidians, according to Regaud and Policard ('03),
possess but a single row of basal bodies located at the surface
of the cell, and the implantation plaque is absent.
DISTENTION O F GLOMERULAR CAPSULES
377
A delicate membrane-like structure which covers the surface of the cell is probably a thin cuticle.
Ureters
Section and cannulation of the ureters did not relieve the
capsular distention, although urine was passed through the
cannulae. No measurements of rate of urine formation were
made.
Blockage of renal tubule
It is evident that the capsular distention is not caused by
a reversal of the ciliary beat nor by obstruction of the ureter.
A third possibility is the partial or complete obstruction of
renal tubules. Such an obstruction might arise from two
causes: first, the nephrostome might sweep into the tubule
not only fluid, but cellular debris from the body cavity; the
latter resulting in obstruction of the lumen. In this case
only the most ventral layer of capsules would be affected,
since only these possess nephrostomes. Secondly, cellular
debris might arise within the tubule itself; thus both types
of capsule might become distended.
Simple experiments made by placing blood cells, carmine
grains, and other small bodies on the ventral surface of the
kidney showed that solid particles were effectively prevented
from entering the nephrostome. The arrangement of the
cilia at a tangent to the axis of the lumen, evident in both
living and fixed material (fig. C), causes at the mouth a
circular movement of the overlying fluid, so that particles on
approaching the vortex of the fluid stream are thrown sharply
to one side. Therefore, tubule obstruction is probably not
caused by debris carried in by the nephrostome cilia.
The funnel-shaped portion of the glomerular capsule opening into the ciliated neck possesses vibratile cilia. The organization of their activity could not be clearly ascertained,
but they also act to prevent or at least to delay blood cells
and other small bodies from entering the ciliated neck from
378
ALFRED M. LUCAS AND H. L. WHITE
the capsule. Such bodies are rotated rapidly and for the
hour or more of observation failed to leave the capsule.
I n fixed and stained serial sections made from kidneys
with abnormally distended capsules the tubular epithelium
was markedly abnormal. The extent of this condition was
traced in individual tubules from capsule to ureter.
Consecutive letters of the alphabet from a t o s have been used t o
label the same renal tubule (pl. 1) at increasing distances from its
origin at the capsule. The capsule is labeled a. The nephrostome, b,
joins the ciliated neck, c. The junction of the neck with the beginning of the proximal convoluted tubule, d, is shown in figure 1. The
portion of the proximal convoIuted tube labeled e lies near the lateral
margin of the kidney. The ciliated junction, f, which connects proximal and distal portions of the tubule is cut longitudinally. Sections
of the distal convoluted tubule cut at various levels of its length
are indicated by letters from g to s. A tubule, x, not the one originating at capsule a, is shown filled with cellular debris near its junction
with the ureter.
The epithelium of the glomerulus, capsule, neck, and
nephrostome appears normal. The proximal convoluted
tubule beginning at its junction with the ciliated neck may
show a complete loss of epithelial cells (fig. 1,d ) . The nuclei
and cells in this case have become rounded, and various stages
in their separation from the basement membrane and desquamation into the lumen may be found. Occasionally a large
area of the tubular surface lacks epithelial cells, the basement membrane being exposed.
The convolutions of the proximal tubule which lie near
the lateral border of the kidney are distended and the epithclial cells are flattened. A cross section of this region is shown
in figure 4, e. I n the upper part of the tubule the cells have
elongated to fill up a gap in the epithelium. Additional examples arc found in the proximal convoluted tubules in the
upper part of figure 1. I n the lower side of the tubule (fig.
4, e) the epithelium is two cells in thickness. The tubule is
cut at right angles to its walls, thus the appearance is not a
sectioning artifact. Flattened pycnotic nuclei similar to those
shown in the elongated tubule below e are occasionally found
DISTENTION O F GLOMERULAR CAPSULES
379
in this region, but they are more numerous in the distal convoluted tubules and collecting ducts.
The ciliated junction (fig. 2, f) between the proximal and
distal convoluted tubules is slightly distended in this particular tubule, which would indicate that the block occurred
distal to this region. The cells appear normal, except for
elevations of the free border, which contain the intracellular
ciliary apparatus, which in this case may be the result of
fixation. This portion of the cell is readily separated from
the rest of the cytoplasm.
The cells of the distal convoluted tubules shown in figures
5 and 6 are still attached to the basement membrane, but
their apical ends are poorly defined. The nuclei bulge into the
lumen. Many nuclei are pycnotic and in j the thinness of the
wall in the lower end of the tubule probably has resulted from
loss of epithelial cells. The same is true of the right side
of m. Many nuclei are darkly stained due to condensation of
chromatin. I n a series of sections from another individual,
the distal convoluted tubules were more severely affected than
in those described. The free borders of the cells were disrupted over large areas, many nuclei were exposed t o the
lumen and sometimes were almost without surrounding cytoplasm. The proximal tubules in this animal were practically
normal.
The tubule, n to s, after leaving the medial border of the
kidney, makes several turns in the region of the capsules.
The beginning of this region, n, is shown in figure 6. The
tubule passes between two distended capsules which compress it to occlusion. Here the passage of fluid would be
greatly retarded if not entirely stopped, and its accumulation
would distend the capsule and that part of the tubule proximal
to the block.
The primary blockage of the renal tubule which produces
the initial distention of a capsule may well result from epithelial desquamation. The only outlet f o r such cells is into
the ureter, and during their passage through the narrower
parts of the tubule, such as the ciliated junction and the distal
380
ALFRED M. LUCAS AND H. L. WHITE
convoluted tubule, there may be temporary stoppages which
start the distention of the capsules. Such a blockage is
shown in figure 3, x. The lumen of this tubule is almost
completely filled with cells. This block occurs at a sharp
bend in the tubule y and x, figure 1,which is a collecting duct
from a capsule other than a.
DISCUSSION
Direct observation of the neck cilia shows that fluid enters
the capsule from the nephrostome against their downward
activity ; this retrograde passage interferes with the coordinated movements of the flagellar portion of the fibrils. Moreover, the cytological picture, in which the structural parts
of the intracellular ciliary apparatus present a directional
and asymmetrical modification coinciding with the plane of
the active beat, renders the existence of reversed ciliary
movements improbable.
The cilia are active when the lumen of the neck is filled with
fluid. I n the sections of living tissue the walls of the neck
may attain a minimum diameter, in which event the cilia are
quiescent, although a definite lumen still persists. It is evident, therefore, that when the glomerulus fails to eliminate
fluid, the neck reacts as if it were contractile. Since it has
been observed in the living sections when the tubule or lower
neck is obstructed that fluid is forced progressively up the
collapsed neck, it follows that the contraction of the upper
neck walls is not due to a negative pressure produced by the
cilia of the lower neck. It remains to be determined whether
elements other than the usual elasticity of cells and basement
membrane are responsible f o r the contracted condition.
Serial sections made from kidneys possessing numerous
abnormally distended capsules revealed degenerative and possibly also regenerative changes in the tuba1 epithelium. Large
numbers of white blood cells are present in the interstitial
tissue-a condition regarded by Drzewina ( '03 and '05) and
Chase ('23) as normal for Proteus and Necturus. Drzewina
and also Dawson ('32) believe that the kidney is a hematopoietic organ.
DISTENTION O F GLOMERULAR CAPSULES
381
The degenerative condition may involve whole cells, as
found in the proximal tubules, o r ceyys may fragment as in
the distal tubules. The numbers lost at one time appear to
be relatively large and they may clog the lumen as shown in
figure 3 , 5 , producing a primary blockage of the renal tubule
resulting in capsular distention_. With partial tubular obstruction one would expect a greater distention, other things
being equal, of the capsules whose tubules possess nephrostomes than of those without them, since additional fluid is
swept in by the nephrostomes. For this reason it is conceivable that in some cases only the primary capsules would
be distended. It is thought that such has been observed.
Fluid accumulates distal to the ciliated neck until the maximum pressure which its cilia can exert, 4.0 to 5.7 cm. water
is attained, after which the intracapsular pres(White, 'B),
sure rises. This occurs all the more readily in primary
tubules which possess a nephrostome. The maximum pressure which the nephrostome cilia can exert is, according to
White, 8 to 11 em. water. Thus, fluid from the body cavity
is forced into the capsule against the action of the cilia in
the neck, as is seen with colored solutions. The collected
contents of such a capsule would be contaminated by fluids
brought in from outside the kidney by the nephrostome and
also by fluid coming back from the tubule. If no nephrostome
exists, the contents of the capsule would probably still be
contaminated, because in the closed capsule-tubular system
ciliary movements produce a circulation and mixing of the
contents of the closed system.
Most of the capsules lie in about the same dorsoventral
plane. The renal tubules pass several times between the
capsules and distention of one capsule might conceivably
affect otbers, because if the distention is sufficiently great,
tubules passing near the capsule may be pinched and flattened,
as shown in figure 6, n. Once this has occurred, flow of fluid
in the second renal tubule is blocked and the capsule connected with this tubule becomes distended also.
382
ALFRED M. L U C A S AND H. L. WHITE
Variatioii in the cytological picture of the renal epithelium
of Necturus has been described by Curry ( 'as),who has indicated the probable sequence in the extrusion process of single
cells and small groups of cells from the proximal tubule.
Undoubtedly, some variations are due to changes in functional activity, as pointed out by Curry, but part may be
seasonal variations accompanying changes of habitat or temperature. It is suggested that the loss of cells from the
epithelium and the markedly abnormal appearance of the
remaining cells are not due to a pathological condition, but
represent transitional stages in reorganization and replacement, although it is recognized that the evidence does not
permit a n unequivocal decision. Animals in which such
kidneys were seen seemed entirely normal in their activities
in the tank and were indistinguishable in their general behavior from those animals in which the kidneys appeared
normal. I n different individuals the severest degenerative
condition was not always in the same part of the tubule,
and throughout the tubules there existed differences in
degree of degenerative and regenerative stages. I f such a
reorganization process occurs, it, is probably progressive
along the tubule. Distended capsules and evidence of reorganization were found in both male and female animals.
It has often been noted in kidneys showing sluggish
glomerular circulation and collapsed capsules when first
exposed, that capsular distention developed after a variable
duration of exposure with no improvement in glomerular
circulation. Three possibilities accountable for this condition suggest themselves ; first, that partial tubular obstruction, existing before the kidney was exposed, permitted passage of the small amount of fluid without resulting in distention; when the kidney surface was flooded with Ringer's, a
large volume swept in by the nephrostome could not pass the
partial obstruction without developing a head of pressure
which resulted in distention. Secondly, that sloughed cells
were present floating in the lumen of the tubule before the
kidney was exposed, but that the flow of fluid was insufficient
DISTENTION O F G L O M E R U L A R C A P S U L E S
383
to produce their aggregation with subsequent obstruction ;
Ringer’s swept in by the nephrostome caused the cells to
lodge at constricted points and sharp turns, resulting in
tubular obstruction. Thirdly, that the cells of kidneys with
poor circulation, whose tubule walls adjacent to the ventral
surface are in early stages of degeneration, would more
readily be injured and dislodged from the basement membrane by exposure and manipulation than the cells of normal
kidneys with rapid circulation. The injured cells become
a n obstruction to the free passage of fluid.
I n this paper it has been pointed out that a distended
glomerular capsule does not necessarily mean a rapid formation of glomerular fluid. The chances that fluid collectsd
from a glomerular capsule may not be normal glomerular
fluid are discussed by White and Lucas ( ’32). It seems probable that the rapid but temporary rate of collectioii of fluid
described by Ekehorn (’31, p. 219) is that of a preformed
accumulation. It would be surprising if some tubular damage
were not caused by the drying process he employed. The
phenomenon of a distended capsule with fluid leaking out
around the site of the puncture, even though a negative pressure of 50 cc. of water was applied t o the pipette, indicates
that the tubule was at least partially obstructed unless the
pipette tip was completely blocked, which was not the case.
It is further felt that his description of a ‘grossly hyperaemic’
glomerulus 011 page 266, which he considers as the type to
be sought for fluid collection, depicts a glomerulus grossly
injured by the current of dry air. It is believed that Ekehorn
was not dealing with normal glomerular fluid at all; if this
belief is correct, his findings as to the composition of
glomerular fluid are valueless, in spite of what appear to be
very valuable contributions to microchemical technique.
The bearing of this retrograde passage of fiuid into the
glomerular capsule on the interpretation of the findings on
glomerular fluid collected from the primary capsules of
Necturus is obvious. I n the earlier work on collection of
glomerular fluid in Necturus this danger was not adequately
T H E ANATOMICAL. RECOIIG, VOL. 5 : ; , X O . 3
384
ALFILEI) M. LUCAS A N D H. L. WHITE
appreciated; in more recent mork this possible source of error
has been circumvented by observiig precautions mhich irislire
against retrograde passage.
LITERATURE ClTEU
CHASE, S. W. 1923 The niesoiiephros and urogenital ducts of Necturus
niaculosis, Rafinesyue. J. Morph., vol. 37, pp. 437-531.
CURRY,L. F. 1929 A eytological study of the proximal and distal tubules of
the uiesonephros of Necturus maculosus. (Undcr norinal and cxperinicntal conditions.) J . Morph. and Physiol., vol. 48, pp. 173-231.
Daivsox, A. B. 1932 Heniopoictic loci in Necturus inaculosus. Anat. Rec.,
vol. 52, pp. 367-380.
DRZEWINA,
A. 1903 Sur lc tissu lymphoide du rein d u Protcus aiiguiiieus Laur.
X o t e preliiuinaire. C . R. Soc. Biol., T. 5.5, pp. 1091-1093.
1905 Contribution i 1'6tude du tissu lgrriphoidc des Ichthyopsidbs.
Arch. 2001. Exper. e t G h . , 4nie serie, T. 3, pp. 14.5-338.
EKEHORX,
G. 1931 On tlie principles of renal function. Acta Medica Scandinavica., Suppl. 36, pp. 1-717.
MCCLUNG,C. E. 1929 Handbook of rnicroscopiral technique. P a u l B. IIoeber,
New York.
PAP~KER,
G. €1. 1903 The reversal of the effective stroke of the labial cilia
of sea aneniones by organic substances. Am. J. Physiol., vol. 11,
pp. 1-6.
KEGBUD, CL., AND A . POLlCARD 1903 Recherches sur l a structure du rein de
quelques opliidiens. Arch. d 'Anat. Micr., T. 6, pp. 191-282.
WHITE, H. L. 1928 Observations on the intrecapsular pressure and the mole
cular concentration of the renal capsular fluid in Necturus. Am. J.
Physiol., vol. 85, pp. 191-206.
1929 Some rneasurenients of ciliary activity. Am. J. Physiol.,
V O ~ . 88, pp. 282-285.
WHITE,11. L., SNU A. M. LUCAS 1932 Observations concerning the collection of
glomerulnr fluid in Neeturus. J. Cell. and Conip. Physiol., rol. 2,
110. 1.
PLATE 1
EXPLANATION OF FIGURES
1 to 6 The lateral edge of the kidney is
the ventral surface is toward the right.
renal tubule a t increasing distances from
another tubule is labeled x t o z.
A more detailed drscription of tlie figures
toward the upper edge of the plate;
The letters, a to s, label tlie saiiie
its origin a t the capsule. P a r t of
is given on page 378 of the text.
PLBTE 1
DISTEKTIOK O F OLOMb’:XbI,AR CAPSULES
AIiPRhD M . LUCAS AND H. 1.
WHIlL
385