oi STANFORD UNIVERSITY PUBLICATIONS
UNIVER SET Y (SERIES

MEDE CAL SCLEN CES

VoLuME I Number 2

A Cytological Study of the Kidney Cell

in Long Continued Hyperfunction with
Relation to Hypertrophy and the
Mitochondrial Apparatus

BY
LUDWIG A. EMGE
Assistant Professor of Obstetrics and Gynecology

From the Division of Obstetrics and Gynecology
Stanford University Medical School
San Francisco

STANFORD UNIVERSITY, CALIFORNIA
PUBLISHED BY THE UNIVERSITY
1921

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No. 1. ARTERIOSCLEROSIS, CARDIOVASCULAR DISEASE; THEIR RELA-

tion To InFEcrious Diseases. William Ophiils, Professor of
Pathology. 102 pp. 10 text figures. 1921. Price, $1.00.

_ No. 2. A Cytotocicat' Stupy or THE KipnEy CELL IN THE LONG
ContinueD HyperFUNCTION wiTH RELATION To Hyprr-
TROPHY AND THE MitocHonpriaL Apparatus. Ludwig A.
Emge, Assistant Professor of Obstetrics and Gynecology.
22 PP. 2 plates. Price, 75c.

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STANFORD UNIVERSITY PUBLICATIONS
UNIVERSITY SERIES

MEDICAL SCIENCES

VotuME I NuMBER 2

A Cytological Study of the Kidney Cell

in Long Continued Hyperfunction with
Relation to Hypertrophy and the
Mitochondrial Apparatus

BY
LUDWIG A. EMGE
Assistant Professor of Obstetrics and Gynecology

\

From the Division of Obstetrics and Gynecology
Stanford University Medical School
San Francisco

STANFORD UNIVERSITY, CALIFORNIA
PUBLISHED BY THE UNIVERSITY
1921

S

CONTENTS

Introduction

- Technique

Results of Vital Staining Studies
Results of Fixed Tissue Studies
Summary

Bibliography

Explanation of Plates

Plates

Cornell University

The original of this book is in
the Cornell University Library.

There are no known copyright restrictions in
the United States on the use of the text.

http://www.archive.org/details/cu31924002903601

A Cytological Study of the Kidney Cell in Long Continued
Hyperfunction with Relation to Hypertrophy and
the Mitochondrial Apparatus

Ever since the knowledge of mitochondria had passed the mere
descriptive stage numerous attempts have been made to interpret their
physiological significance. This investigation was undertaken in order to
determine whether wide variations in the duration and degree of experi-
mentally induced renal activity were associated with any demonstrable
peculiarities in the mitochondrial content and general cellular structure of
the renal tubules. Part of the tissues for this study were obtained through
the courtesy of Dr. Addis and Mr. Shevky of the Division of Medicine,
who have kindly contributed the life history of the animals used.

NOTE BY T. ADDIS AND A. E. SHEVKY ON THE LIFE HISTORY OF
THE MATERIAL DEALT WITH

(From the Division of Medicine)

There is no direct method by which the “work” of the kidney can be measured,
but changes in rate of work may be assumed to be indicated by changes in rate
of metabolism. Barcroft(1)* has shown that an increased rate of urea excretion
is associated with an increased consumption of oxygen by the kidney, although
increases in sodium chloride and water excretion are not accompanied by any
appreciable change in gaseous metabolism. On these grounds one would expect to
be able to produce renal hypertrophy in intact animals by increasing the work of
their kidney by constantly and over a long period giving them large amounts of
urea in their food, At least such a result might be expected if the assumption is
correct that the increase in size of the remaining kidney after unilateral nephrectomy
is due to the additional work it has to perform.

Twenty-four newly-weaned white rats about a month old were divided into
two groups as nearly as possible alike in regard to the size, weight, and ancestry
of the individuals of which they were composed. Each group was kept in a separate
cage of the type described by Robertson(2). The diet used was one shown by
Osborne and Mendel(3) to be adequate for growth and maintenance. It contained
15 gms. of lard, 33 gms. of cornstarch, 5 gms. of agar-agar, and 43.5 gms. of
powdered milk. In order to facilitate the combination of these ingredients into a
homogeneous paste into which urea could be easily incorporated we added 50 cc.
of whole milk for every 100 gms. of the combined foods. Urea in substance was
rubbed in a mortar into that part of the food given to the urea-fed rats, and in
order to obviate any possibility of error a little charcoal was added at the same
time. For the first two months 5.5 gms. was given with each 100 gms. of food,
during the third month 8 gms., for the fourth and fifth months 11 gms., and so on
with successive increases until from the 13th to the 16th month, when the experi-
ment was terminated, 17 gms. of urea was taken in every 100 gms. of food. There

* Figures in parenthesis refer to bibliography on pages 121 and 122.

108 A CYTOLOGICAL STUDY OF THE KIDNEY CELL

was no limitation of water intake, except that with the idea of straining the urea-
concentrating capacity of the kidney, we removed the water tube from the cage of
the urea-fed rats for one hour each day following the giving of fresh food.

There can be no doubt that by these measures we succeeded in making the
kidneys of the urea-fed group do much more work than those of the controls. In
spite of repeated efforts, however, we cannot give any accurate measure of the
difference, since we did not succeed in entirely overcoming the technical difficulties
of collecting urine from such small animals. We did find, however, that the urea-
fed rats took about twice as much water and excreted about twice as much urine.
This urine at times contained as much as 10 per cent of urea. The funnel over
which the urine ran into the collecting vessel was usually coated with urea crystals.
Examination of the feces showed that urea was not excreted by the gastro-
intestinal tract.

At the end of sixteen months various accidents had reduced the number of
rats to eight, four controls and four urea-fed. After a functional test, which will
be described below, these rats were killed by bleeding from the carotid artery after
very short ether anaesthesia, and the kidneys at once removed and weighed. The
results are given in Table I. They fail to show the existence of any hypertrophy.

TABLE I
KIDNEY WEIGHTS OF CONTROL AND UREA-FED RATS
CoNTROLS
Kidney Weight asa
Percentage of Body
No. Kidney Weight Body Weight-Alimentary Tract Weight-Alimentary Tract
gms. gms. per cent.
1 1.37 182 0.75
2 0.93 124 0.75
3 1.13 132 0.85
4 1.30 132 0.99
Urea-Fep
Kidney Weight asa
Percentage of Body
No. Kidney Weight Body Weight-Alimentary Tract Weight-Alimentary Tract
gms. gms. per cent.
5 1.27 148 0.86
6 1.01 115 0.89
7 1.40 152 0.93
8 1.06 103 0.97

On the day before each animal was killed an attempt was made to measure
the urea-excreting capacity of the kidneys. In order that both groups might be
under the same conditions, no urea was given to the urea group for two days
before the experiment. Urea in concentrated solution, 0.5 gms. for one pair of
rats, and 0.375 for the other three pairs, was injected from a syringe through a
fine tube into the stomach. The rat was then placed over a funnel, so that all
urine was collected. After three hours the carotids were cut under ether anaes-
thesia, and the blood collected. The urea content of both blood and urine was
determined by the usual urease aeration method. The results are given in Table II.

INTRODUCTION 109

TABLE II
UREA-EXCRETING FUNCTION OF CONTROL AND UREA-FED RATS

ContTROLS
No. Urea in One Hour's Urea in 100cc Ratio: Urea in Urine
Urine Blood Urea in Blood
mgs. mgs.
1 75 165 0.45
2 48 186 0.26
3 34 76 0.34
4 8 132 0.06
Urea-Fep
No. Urea in One Hour’s Urea in 10Ccc Ratio: Urea in Urine
Urine Blood Urea in Blood
mgs. mgs.
5 85 102 0.83
6 44 93 0.47
7 64 140 0.46
8 58 60 0.97

The higher rate of urea excretion in spite of the lower level of blood urea
concentration in the urea-fed rats seems to indicate a greater facility in the excre-
tion of urea, in spite of the absence of any appreciable increase in the size of their
kidneys. But the number of experiments is much too small to allow for any
definite conclusion.

The two groups of animals whose physiological history has been de-
scribed by Addis and Shevky were supplemented with another two groups
of white rats similar in age and weight. One group was fed general food
and the other was given urea food as noted in the life history of the tissues
of the first two groups. After several days’ feeding, the first pair, urea-
fed and control, was killed. Then within two weeks a pair was killed
every other day. The idea was to obtain tissues for study of possible
early changes in the renal cellular structure produced by urea feeding for
a comparison with tissues from kidneys in a normal state of activity.

There were, therefore, four distinct groups available for comparative
study; the first, composed of kidneys which since early youth had been
subjected to a constant strain in the excretion of large amounts of urea;
the second, a group which had been fed a similar diet without urea but
had been given a single dose of urea just before death; the third, a group
which was fed urea over a period of from two to fourteen days before
death; and the fourth, a group fed on ordinary diet which received no
urea whatsoever.

110 A CYTOLOGICAL STUDY OF THE KIDNEY CELL

TECHNIQUE

Owing to the fact that the kidneys had to be weighed for reasons
explained in the above life history, fixation by arterial injection could not
be practiced. This would in any case have been impracticable since it
would not have permitted vital studies.

As fixing agents Bensley’s(4) formalin-zenker, Regaud’s(5) neutral
formalin-bichromate, and Kolster’s(6) chromalum and fluorchrome mix-
tures were used. Controls were also fixed in chemically pure, freshly neu-
tralized 10 per cent formaldehyde. It was impossible to obtain osmic acid
at the time of this investigation (1918). Tissues in pieces of 2 mm. thick-
ness were fixed in these agents from one to three days and then chromi-
cized from three to nine days. A temperature of about body heat was
maintained throughout the period of fixation. The fluids were changed
daily. The fixed material was cleared with either cedar oil, bergamot oil,
or xylol. I have found practically no difference in the shrinkage of the
tissues with either of the three, and therefore have used xylol in prefer-
ence as it is the fastest and least expensive of all clearing agents. Paraf-
fin, 60°, was used as the embedding medium. Sections were cut serially
and about 5 uw thick. Altmann’s(7) anilin-fuchsin picric acid method in
its original form and as modified by Cowdry(8) and Galeotti(9), Bens-
ley’s(4) anilin-acid-fuchsin methyl green, Bensley’s(4) copper-chrome-
hematoxylin and Heidenhain’s iron-hematoxylin were used. Simple con-
ventional stains were employed as a check on the general picture. Ap-
parently it is not necessary to use osmic acid to obtain good fuchsin pic-
tures. Such simple bichromate mixtures as mentioned above will pre-
serve mitochondria excellently and allow good staining results.

Among the fixatives the formalin-zenker and the neutral formalin
bichromate mixtures with a period of chromization have given by far the
best results. Of the staining methods the anilin-acid-fuchsin methods
are quicker but otherwise in no way superior to the other methods
mentioned.

Fresh tissues were studied in a normal saline solution of 1: 10,000
Janus green 6. Arnold’s(10) neutral red and methylene blue were used
in several control animals. Janus green studies of renal tissues are very
difficult. First of all, the dye seems to penetrate very slowly, and one is
therefore not certain whether the mitochondria have not changed in ap-
pearance by the time they stand out plainly; secondly, mitochondria are
usually massed so densely in the convoluted tubules that a clear picture is
very rarely obtained; thirdly. in order to obtain thin enough pieces of
tissue one must either tease them out or cut them with the freezing micro-
tome. Either method furnishes ground for controversy. It seemed to
me, however, that sections cut with a good freezing outfit are preferable
to teased-out tissues.

RESULTS OF VITAL STAINING STUDIES 111

RESULTS OF VITAL STAINING STUDIES

Nothing remarkable was found in the vital staining studies. It
was at once plain that in spite of the poor penetration of the dye, the mito-
chondria of the convoluted apparatus by far outnumbered those of the
remaining tubular structure. Comparing controls to urea-fed rats of the
first group, there was no striking difference in the mitochondrial picture
in general. In all tissues studied the impression was gained that in the
control rats the mitochondria of the convoluted tubes were arranged in
distinct, closely packed stout rods or in bands of medium-size granules,
which extended about two-thirds into the cell and which were most nu-
merous near the base. In the collecting apparatus the finer, less numerous
granular mitochondria were distributed throughout the cell, leaving a
clear space around the nucleus. In the large tubules of Bellini the stout
rodlike form appeared again, but the number was few, and I judged that
more than half of the cells were mitochondria-free. In the urea-fed rats
the only difference in the mitochondrial picture was that the granular
form in general prevailed, but position and arrangement seemed to be the
same as in the controls. At any rate, no such morphological differences
existed as would warrant inferences as to differences in secretory activity
in the proximal convoluted tubules of the four groups of kidneys.

The use of vital color reactions to solve the secretory problem of the
kidneys dates back to 1874, when Heidenhain(11) and Neisser first in-
jected dyestuffs into animals and found them in the convoluted, tubules,
while the glomeruli and capsules were free from color materials. This
work in general has been substantiated by a number of investigators.
Since the exhaustive histochemical studies of Leschke(12), confirmed by
Oliver(13), it seems more clear than ever that urea and perhaps other
urinary solids are secreted (or excreted) in the convoluted apparatus,
while the glomerular apparatus is primarily concerned with the secretion
of practically solid-free fluids. The difficulty enters when one tries to
obtain evidence by vital dye studies as to whether the mitochondrial ap-
paratus is directly concerned with the secretory mechanism of the cell
of the convoluted tubule. One must not forget that most of the dyestuffs
used in histological methods are of colloidal nature, and consequently
when seen in cells that secrete substances crystalloid in nature they cannot
be used as an absolute criterion or parallel. The presence of dye mate-
rials in the renal cells brought there by the blood or lymph stream must
be considered a physical rather than a chemical reaction, as was brought
out by Suzuki(14) working in Aschoff’s laboratories. He found that the
dye appeared in the urine long before the granular structures of the con-
voluted tubules showed any signs of staining. Carmine in various con-
centrations was used and at an “early stage of excretion” many finely
granular carmine casts were present in the collecting tubules. Suzuki

112 A CYTOLOGICAL STUDY OF THE KIDNEY CELL

states that the presence of these granules does not point to carmine-
stained secretory granules derived from the epithelium of the convoluted
tubules, but rather to a product of precipitation of the dyestuff in solu-
tion, which is only in part passed through the convoluted tubules, while
some of it passes through the glomerular apparatus. He calls attention to
the fact that by pushing the carmine injections there is a gradual accumu-
lation of dyestuff in the renal epithelium which ultimately leads to stain-
ing of the true cell granules. The latter, therefore, must be sharply differ-
entiated from carmine granules. His results lead him to believe that the
carmine-absorbing granules have nothing to do with the actual secretion
of the dye material.

The objection that might be raised is that carmine is not a specific
vital dye for mitochondria. Nevertheless the fundamental result remains
unchanged. Many investigators are in accord that the striations of the
convoluted tubules of the kidney are homogeneous rods when seen in
fresh tissues, but become granular in appearance under various condi-
tions. Some have considered this a true secretory phenomenon, others an
artefact. Arnold(15) thinks that the individual granules of the living
renal epithelium are pre-existing in the rods and joined together by seg-
ments. In my studies I have repeated Arnold’s neutral red and methy-
lene blue method, and I am convinced that such a differentiation can be
obtained. The granules are stained red and the segments blue. There
are, however, certain objections. First of all, neither dye is specific for
mitochondria; secondly, in order to obtain a good differentiation of the
binding segments penetration has to be prolonged, and it is well known
that mitochondrial rods will break up slowly into granules if they are
exposed to changes in temperature or to substances poisonous to tissues.
This was shown by Lewis and Lewis(16) in tissue cultures, by Suzuki
in his carmine injections, and in various pathological conditions bv
Ophiils(17), Takaki(18), Israel(19), Burmeister(20), Landsteiner(21),
Stork(22), Pfister (23), and others.

While Janus green is considered a specific vital dye for mitochondria,
it is not a very satisfactory dye for the kidney as it penetrates very slowly,
even under the most favorable conditions, as on a warming stage. It has
the tendency to bring out a granular rather than a rodlike structure, and
the observation made here that the granular structure was seen more
often in the urea-fed rats may be the’result of various circumstances. If
no warming stage is used the difference in temperature alone may be
sufficient to produce granular dissolution. If a stage is employed such
changes in osmosis as may be produced by evaporation may create granu-
lar changes. Césa-Bianchi(24) has protested against the assumption of
pre-existing granules on the basis that unstained fresh tissues have solid
rods. He maintains that the granular picture is created by fixation or by

RESULTS OF VITAL STAINING STUDIES 113

the influence of foreign substances. It did occur to me that by polariza-
tion such a question could be settled. Unfortunately, at the time of this
investigation this chance was overlooked.

The only result in this part of the investigation that can be offered
as a fact is, that in Janus green studies the tubules which were proven
by Leschke(12) and Oliver (13) to give off urea also are the ones that
have the greatest abundance of mitochondria, and that the tubules which
Suzuki(14) considered the resorption tubules, have no or very little mito-
chondrial structure. But that there is a difference in vital staining reac-
tion between the mitochondria of kidneys after long-continued or acute
strain in urea excretion, as compared with those acting normally, cannot
be asserted.

Since in some of the first tissues studied vitally there were seen
spherical, highly refractile bodies of various sizes apart from mitochon-
dria, Sudan III and scarlet r. were employed in the remaining tissues
after a Janus green penetration had been obtained. These round bodies
stained brilliantly and instantaneously with the so-called fat dyes which
did not influence the green-stained mitochondria. The arrangement of
these bodies was irregular and without any apparent similarity in neigh-
boring cells in the entire group of tissues studied. That is to say, they
were as numerous in the controls as in the urea-fed rats of the first and
second group. From this I judge that normally there is free fat, or a
substance related to fat, in the cells of the kidney of white rats. At no
time did these round bodies stain with Janus green, and this dye is specific
for mitochondria. It is obvious that these bodies are not aberrant physi-
cal forms of mitochondria. In the fixed sections similar, or perhaps the
same, structures have been seen by several investigators, and explanations
have been offered which point out that these bodies may be concerned
with secretion. Heidenhain(25), Hoeber and Koenigsberg(26), Van der
Stricht(27), and Gurwitsch(28) have described intracellular vacuoles of
various positions and sizes. These vacuoles were thought to act as collec-
tors of secretory materials. Gross(29) and Hirsch(30) were not able to
confirm the presence of these vacuoles. Retzius(31) observed these struc-
tures but did not think them vacuoles. He rather interpreted them as true
droplets of secretion. In these droplets he saw very fine granules con-
nected by fine threads. At a time when the cell appeared high and the
brush border had disappeared these droplets were supposed to be expelled
into the lumen of the tubule.

In my fixed sections the position and sizes of the occasional “‘vacu-
oles” observed seemed to correspond in location to the fat bodies de-
scribed above. At no time could I find sufficient proof that in the vitally
stained tissues fat droplets were similar in arrangement or staining to
mitochondria. Nevertheless, it may be possible that these substances are

114 A CYTOLOGICAL STUDY OF THE KIDNEY CELL

derived from mitochondria jas pointed out by Ophiils(17), but if that is
so it is evident that some change has occurred involving radical altera-
tion in the staining affinity of the mitochondrial substance.

RESULTS oF Fixep TissugE STUDIES

In studying the low-power picture the cortex stands out massively
on account of the mitochondrial richness of its tubules. This picture
varies in its color intensity with the variations in mitochondrial density
and also with the variations in fixing agents. Nevertheless, the color
coefficient between medulla and cortex remains stationary. On viewing
the cortex, even with the lowest power, one can easily distinguish the
proximal convoluted tubules on account of their greater staining intensity.
Fig. 1 gives a fair demonstration of the difference in color density between
proximal and other tubules of the cortex.

In comparing sections from the four groups of kidneys differences
could always be found, but further study invariably showed that such
differences also existed within the individual group and therefore could
not be used in differentiating the groups. Only the study of serial sections
can protect against unwarranted conclusions. As a demonstration I wish
to cite an observation which, without serial study, would easily have led
to faulty inferences, if coincidence should have thrown the observations
mostly into one group of tissues.

In the studies of the group described by Dr. Addis a peculiar picture
of divisional action of groups of convoluted tubules is observed, if one
may use this term for the sake of simplicity (see Figs. 2 and 3). It is
at once apparent that in some tissues entire groups of convoluted tubules
with their connecting segments (Schaltstiicke) stand out very plainly by
virtue of their mitochondrial concentration in contrast to other neighbor-
ing groups, which have less and fainter staining, but otherwise similar
appearing mitochondria. A casual observer might be easily misled by
such a picture. In this study it seemed at first that such a grouping was
more often seen in the tissues of the rats which had undergone prolonged
urea feeding. But the study of a large number of sections from all the
groups dissipated this impression. All kidneys showed this peculiarity in
some part or other of their structure. While this phenomenon does not
give any help in differentiating the kidneys of urea-fed from control rats,
it is of interest in relation to Lindemann’s(32) hypothesis of a division
of labor in the individual kidney in which certain groups of tubules sup-
posedly are allowed to rest while others are active. At any rate it illus-
trates the fallacies of drawing conclusions from sections which are not
representative of the entire organ.

The absence of any appreciable increase in the weight of the kidneys
in Group I, which had been fed urea over a long period, precluded the
existence of any gross difference in the number and size of the cells as

RESULTS OF FIXED TISSUE STUDIES 115

compared with the second group. Yet, it was thought possible that there
might be a constant difference in the minute structural arrangement with-
in the cell, and a careful comparison was therefore made in order to de-
termine whether such qualitative distinctions might not be associated with
the various degrees in urea feeding, especially as compared with the group
which did not receive any urea at all.

In studying the general cell picture one finds that the height of the
cell of the proximal convoluted tubule is fairly constant, and such varia-
tions as exist seem to depend somewhat on the mitochrondial content of
the cell. If the content is low the icell cupola is high and the lumen is
very narrow (see Figs. 6 and 7). This is especially true when the mito-
chondrial apparatus is in a state of advanced granular dissolution (see
Fig. 8). With the increase in the numerical content of the mitochondria
the cell becomes somewhat lower. The cell cupola at once becomes clear
and the plasma just at the base becomes transparent. The more definitely
the mitochondrial apparatus assumes the “batonné” arrangement the
more apparent becomes this decrease in cellular height (see Fig. 11).
With this go certain variations in configuration and width of the lumen.
When the mitochondrial content is low and the plasma is dense through-
out the cell the lumen is narrowest and somewhat stellate in character
(see Figs. 6 and 7). As soon as the granular structures become definite
in their arrangement, the lumen widens and only fine strands of proto-
plasmic material bridge the lumen in an irregular fashion (see Fig. 12).

In comparing the cells of the tubular structure of the kidneys of the
various groups it was at once apparent that those from animals fed urea
did not show any evidence of a distinctive increase in the size of the cells
of any part of the tubular apparatus. In any kidney from any group it
was possible to find the lesser degrees in variations in the size of individ-
ual cells which have been discussed above (see Figs. 9 and 10). One may
therefore conclude that urea feeding does not stimulate hypertrophy of
the cell of the kidney even if such feeding is carried to the extreme by
extending it over the life period of the animal in question.

Also the appearance of the brush border does not offer any conclu-
sive evidence that it is influenced in any way by the forced feeding of
urea. I cannot agree with those writers who think that the variations
in the morphological appearance of the brush border are indicative of
phases in the secretory process. By some it is claimed that it gradually
rises, due to cellular pressure, until it is thrown off to allow the secretory
material to enter the lumen. Others do not share this view, and consider
the passage of secretion material as a true secretion phenomenon. Espe-
cially Sauer(33) in his earlier studies opposed the former idea, since he
always found a typically striated appearance of the brush border at any
stage of secretion. Of late Kolster(6) once more has drawn attention to

116 A CYTOLOGICAL STUDY OF THE KIDNEY CELL

the brush border. He believes that in what he calls the “resting stage”
(when the cells are high and the lumen narrow) the brush border is low
and possesses a very distinct striation, while in the active stage the brush
border increases in thickness and appears indistinct in its markings.
Whatever may be the significance and affiliation of the brush border
with other cell structures, it undoubtedly is one of the finest architectural
tissue structures and therefore exposed to the greatest variations in fixing
artefacts. In these studies this was confirmed by the careful examination
of pieces of tissue where for unknown reasons penetration had lagged.
Here, in sections cut deeper in the piece of the tissue, the brush border
was severely damaged and often had a “glued-together” appearance.
Such a condition may easily lead to misinterpretations. And as coinci-
dence occasionally will produce such an artefact in a group of tubules
where the mitochondrial content is low, it is easy to associate the two and
regard them, as Kolster(6) does, as indicative of a distinct phase in se-
cretion. I admit that there are differences in the thickness of the brush
border, and this is regulated somehow by the condition of the plasma of
the cell of the convoluted tubule, since the brush border appears clearest
whenever the cell cupola is most transparent. This is true of vitally
stained as well as fixed tissues. Just because it is so, it may be an optical
illusion, because of the fact that more light is permitted to strike the
brush border from all sides under this condition, while it can strike the
brush border only from three sides when the plasma is dense, and there-
fore the striations may not be seen as clearly. Consequently the distinct-
ness of the brush border striations must vary normally. The actual
height of the brush border varies very little within the individual section
of the proximal tubule. In well fixed tissues I have never seen a place
where the brush border was shed or being lifted off. I am unable to pre-
sent any photomicrographic proof for this statement, as I have not suc-
ceeded in taking a picture which will show the finer inner cell structure
clearly and at the same time do justice to the brush border. In fact, with
the mitochondrial stains brush-border photography is almost impossible.
Occasionally one sees granular structures in the brush border (see
Figs. 4, 8,12). They take the same stain as do the granular structures
of the cell itself. These granular bodies, which vary greatly in size and
which are commonly larger than mitochondria when there is a dissolu-
tion of the rods, may constitute a direct or indirect product of the mito-
chondrial apparatus and merely represent a physical combination of more
than one of the large granules seen in the cell cupola in certain phases
in the life cycle of the cell of the convoluted tubule, or, in fact, in any of
the cells of the tubular structure in general (see Fig. 8). Only occasion-
ally are similar granules seen in the cell lumen or within castlike
formations. Also, here they do not differ morphologically from the cell

RESULTS OF FIXED TISSUE STUDIES 117

granules. To call them mitochondrial secretion products offhand is open
to criticism, since we have not accepted it as an absolute fact that the
mitochondria of the kidney enter into the secretory role.

When we now compare the various groups of the kidneys studied in
reference to the appearance of their brush borders, it again must be ad-
mitted that there is no proof that this structure is influenced in any way
by urea feeding. Such variations as may be found can be demonstrated
within each individual group, and therefore must be considered as a nor-
mal variation of the brush-border picture.

In the studies of the mitochondrial apparatus of the kidney of the
white rat certain generalities have been agreed upon by all investigators.
Upon general survey one is always impressed with the intense staining of
the proximal and to a lesser degree of the distal convoluted tubules. In
the proximal tubules the mitochondrial apparatus is very distinct and con-
sists of true rods or of varying degrees of granular dissolution of these
rods. The mitochondrial numerical concentration is higher here than in
other segments of the tubule. Also the affinity of the individual granule
for certain stains is greater because the granule is larger here than in any
other segment (see Figs. 4 and 5). Both conditions explain the color
prominence of the proximal and, to a lesser degree, of the distal convo-
luted tubules. The greater staining affinity is aparently of purely physical
and not chemical origin, this assumption being based on a greater concen-
tration of the mitochondrial material in one unit. In the medial and dis-
tal parts of the proximal tubules the mitochondrial structure is less dense
and the granular dissolution more pronounced. The descending limb of
the loop of Henle is very poor in granules and occasionally appears free
from them, so that it is hard to recognize them with mitochondrial stains.
the chainlike or rodlike arrangement of the mitochondria, which is so
typical of the proximal convoluted tubules, is suggested again in the mito-
chondrial arrangement of the ascending limb of Henle’s loop. But here
the granules are very much more scarce, smaller and quite irregularly
arranged. Especially in the cell cupola this irregular arrangement is ap-
parent and gives to it a peculiar, loose appearance. The distal convoluted
tubules are similar to the proximal convoluted tubules, except that the
mitochondrial apparatus is less dense and the cell is lower in general.
The epithelium of the collecting tubule is very low and the granular struc-
tures fine and well scattered (see Figs. 13 and 14).

Studying on this general basis the kidneys of the rats which had
undergone prolonged urea feeding, and comparing the results with those
of the rats of the other three groups, no essential difference in the mito-
chondrial apparatus can be detected. Also in this group the intensely
staining mitochondria of the proximal convoluted tubules dominate the
picture (see Figs. 4, 5, 9, 10, 12). But on careful study one is at once

118 A CYTOLOGICAL STUDY OF THE KIDNEY CELL

impressed with the want of constancy in the appearance of the mitochon-
dria in different or even in the same microscopic sections from the same
kidney. This holds good also in the other groups. Serial sections prove
that in every kidney distinct variations in the structural picture of the
mitochondria of the same proximal convoluted tubule are always present ;
that is to say, the variations depend upon the level at which the section
is cut. There is always a gradual transition in the shape of the mito-
chondrial structure from one segment of the tubule to the other. Even
within the central limit of the segments studied such variations are pres-
ent. Then again neighboring segments of the same type but of different
tubular units may vary distinctly in the structural arrangement of their
mitochondria as to size, number, and shape. But such variations cannot
be the result of urea feeding, since they are also found in the kidneys
of the rats that were not fed any urea at all. There are about as many
variations as there are kidneys studied, and I am afraid one could carry
the descriptions of the various combinations of these on to the infinite.
True enough, on a general quick survey of the sections of the group
that received no urea, the picture of the kidney of the white rat described
for the normal, holds good, but when it comes to comparing even neigh-
boring sections of the same tubular group, variations are also found here
at once. For instance, there is a distinct difference in the granular disso-
lution in Figs. 8 and 12, which are sections from the controls of the first
group. But the same is true of the kidneys of the rats fed urea over a
long period (see Figs. 6, 9,10). Then again the dogmatic picture which
has been described for the normal is represented by Fig. 11, but it is a
picture of the proximal convoluted tubule from a kidney exposed to a long
urea strain. Here the beautiful “batonné” arrangement is very apparent
and dominates the optical impression received. These instances merely
illustrate the point raised.

In comparing the individual mitochondrial granules of the various
groups no hypertrophy was observed in those where urea had been fed,
and when compared to controls from the first and second group no nu-
merical increase could be established. It is impossible to segregate the
various groups of rats by the appearance of their mitochondrial apparatus.
Granular dissolution or breaking up of the solid rods is not any more a
characteristic of the urea kidneys than of the control kidneys (compare
Figs. 6 and 7,9 and 12). Nor is the size of the granules produced dur-
ing dissolution any indication that the mitochondrial apparatus of the
proximal convoluted tubules is in any way affected by increased urea
secretion, since the same picture is found in controls that did not receive
any urea whatsoever. If we pay attention to the qualitative difference in
staining’ it can be stated that there is none except for the usual normal
variation.

RESULTS OF FIXED TISSUE STUDIES 119

The picture of the mitochondrial structure of the other tubular seg-
ments corresponds in general to those described for the normal with cer-
tain small variations under ordinary conditions. The glomeruli and their
capsules are practically free from mitochondria in the kidney under normal
activity, and urea feeding does not bring on an increase (see Figs. 15 and
16). There are no indications anywhere in the tissues that pathological
changes had been produced by urea feeding.

A number of observers have ascribed a secretory role to the mito-
chondria of the kidney, such conclusion being based on animal experi-
mentation. For instance, during hibernation Ferrata(34), Disse(35), and
Monti(36) found the following changes from the normal under ordinary
circumstances: the lumina of the convoluted tubules are narrow and the
epithelium is not sharply outlined but markedly granular. Near the base
the granules assume a regular chainlike arrangement, while the inner and
less dense part of the cell is traversed by fine threads. When secretion
sets in, this latter part of the cell becomes quite transparent, bulges toward
the lumen and granules replace the threads. This appearance was con-
sidered a storage of granules supposedly characteristic for a prophase of

_ , secretion.

Also Takaki(18), working with starvation and dry feeding, and
Kolster(6), using similar methods together with sodium chloride injec-
tions, concluded that mitochondria have a definite place in the secretory
process of the kidney. Their investigations tend to divide the stages of
secretion according to the appearance and arrangement of mitochondria.
Their findings have been confirmed by Schultze(37). It is of greatest
importance that Regaud(38), after most exhaustive studies, concluded
that all the various types of granules, rods, and filaments described under
different names belong to the same group, namely, mitochondria, and that
they varied in their appearance according to a number of conditions
among which were changes in the secretory activity of the cell. It was
Simon(39), Retterer(40), and Henschen(41), and to some degree
Dumas(42), Mayer and Rathery (43), who substantiated Regaud’s con-
clusion by further experimental work. Nevertheless, the changes men-
tioned have been thought to be artefacts by Prenant(44), Policard(45),
Levi(46), and Ciaccio(47).

I cannot agree with the latter, since 1 feel certain that the mito-
chondrial play seen in these studies, and of which photomicrographs are
presented here, is not the result of variations in fixation. It is true that
different fixations (fixing agents) of pieces from the same tissue will show
slight differences in the appearance of the individual mitochondrial unit,
but aside from this the variations observed within one piece are constant
for that fixation, and correspond to the variations in pieces fixed differ-

120 A CYTOLOGICAL STUDY OF THE KIDNEY CELL

ently, after one has allowed for the deduction of such changes as are
produced by the fixing agents generally.

Leschke, and later, Oliver, found that urea was secreted in the prox-
imal convoluted tubules. The mitochondrial concentration is highest in
this part of the tubular apparatus, and the mitochondrial changes, which
by many have been thought to be of secretory importance, are also here
most pronounced. It is, therefore, only natural to attempt to link together
these observations. But the studies cited here lead me to believe that urea
feeding of various degrees does not influence beyond normal variations
the physical appearance of the mitochondrial apparatus of the kidney of
the white rat in general and of its proximal convoluted tubules in par-
ticular.

SUMMARY

The result of an anatomical comparison between the kidneys of white
rats on a mixed diet, calling for no unusual renal activity, and the kidneys
of rats which for prolonged or brief periods of time had undergone a
markedly abnormal strain in the excretion of large amounts of urea, may
be summed up as follows:

1. Prolonged urea feeding led to no increase in the size of the cells
of any part of the glomerulo-tubular apparatus.

2. Neither a prolonged nor a brief increase in urea excretion had any
observable effect on the appearance of the brush border.

3. A careful study of the mitochondrial apparatus of the kidney
failed to show that variations in the rate of urea excretion during life
were associated with any change in the number, size or position of the
mitochondria.

I take occasion to express my sincere appreciation to Dr. T. Addis
for his most helpful suggestions in editing this paper, to Miss Ella Wing
for her splendid help in the technical work, and to Dr. F. E. Blaisdell for
his kind advice in photographic difficulties.

SraNnForp University HospiraL, SAN FRANCISCO

On nun & WH

BIBLIOGRAPHY 121

BIBLIOGRAPHY

. Barcroft, J., and Brodie, T. G.: Journal of Phystol., 33, 52, 1905-6. London.

. Robertson, T. B., and Ray, L. A.: Journal of Biol. Chem., 24, 347, 1916.

. Osborne, T. B., and Mendel, L. B.: Journal of Biol. Chem., 17, 401, 1914.

. Bensley, R. R.: Amer. Jour. Anat., 12, 297, 1911.

. Regaud, C.: Arch. d’ Anat. micr., 11, 291, 1910,

. Kolster, R.: Ziegler’s Beitrage, 51, 209, 1911.

. Altmann, R.: “Die Elementarorganismen, etc.,” p. 27. Leipzig, Veit Co., 1890.
. Cowdry, E. V.: Contributions to Embryology, Carnegie Institution, Washing-

ton. No. 11, p. 27, 1916.

. Galeotti, G.: Intern. Mtschrft. f. Anat. u. Phys., 12, 461, 1895.
. Arnold, J.: Virchow’s Archiv., 169, 1, 1902.

. Heidenhain, R.: Arch. f, micr. Anat., 10, 1, 1874.
. Leschke, Erich:

(1) I’ter Kongress fiir innere Medizin, Wiesbaden, 1914, p. 635.
(2) Zeitschr. f. Klin, Medizin, 81, 14, 1914-15.

. Oliver, Jean: Jour. of Experimental Medicine, 23, 301, 1916.

. Suzuki, T.: “Zur Morphologie d. Nierensekretion.” Jena, 1912, Gustav Fischer.
. Arnold, J.: “Weber Plasmastrukturen.” Jena, 1914, Gustav Fischer.

. Lewis, M. R., and W. H.: Am, Jour, of Anat., 17, 339, 1915.

. Ophils, W.: Proc. Soc. Exp. Biol., 4, 135, 1907.

. Takaki, K.: Arch, f. mikr. Anat., 70, 245, 1907.

. Israel, O.: Virchow’s Arch., 123, 310, 1891.

. Burmeister, Th.: Virchow’s Arch., 137, 405, 1894.

. Landsteiner, K.:

(1) Wiener klin. Wochschft., 41, 1904.
(2) Ziegler’s Beitraege, 33, 157, 1903.

. Stork, O.: Sitzbr. d. k. Akad. d. Wissenschaften, Wien., 115, Abt. 3, 1906.
. Pfister, M.: Ziegler’s Beitraege, Arnold’s Festschrift, 37, Suppl. 7, p. 525, 1905.
. Césa-Bianchi, D.: Intern. Mtschrft. f. Anat. u. Phys., 27, 89, 1910.

. Heidenhain, M.: “Plasma und Zelle,” Jena, G. Fischer, 1911.

. Hoeber, R., and Koenigsberg, A.: Pflueger’s Arch., 68, 323, 1905.

_ Van der Stricht, O.. Amnnal. d. l. Soc. d. Med. Gent., 1892, Reprint.

. Gurwitsch, A.: Pfliiger’s Arch,, 91, 71, 1902.

. Gross, W.: Ziegler’s Beitr., 5Y, 528, 1911.

. Hirsch, C.: Anat. Hefte, 41, 131, 1910.

. Retzius, G.: Biologische Untersuchungen, 17, 53, 1911.

. Lindemann, W.: Ergebn. d. Phys., 14, 643, 1914.

. Sauer, H.: Arch. f. mikr. Anat., 46, 109, 1895.

122 A CYTOLOGICAL STUDY OF THE KIDNEY CELL

34. Ferrata, A.: Arch. ital, Anat. e embr., 4, 505, 1905 (see Schwalbe’s Jahres-
berichte for 1905, p. 505).
35. Disse, J.: Anat. Hefte, 2, 143, 1892.
36. Monti, R., and A.: Mem. let. ital. d. Istitut. Lomb. Pavia, 14, 82, 1900.
37. Schultze, O.: Anat, Anz., 38, 257, 1911.
38. Regaud, Cl.:
(1) C. R. Soc. Biol., 64, 1145, 1908.
(2) C.R. Soc. Biol., 65, 206, 1908.
39. Simon, Ch.: Compt. Rend. Soc. Biol., Paris, 50, 443, 1898.
40. Retterer, E.: Compt. Rend. Sac, Biol., Paris, 60, 560 and 611, 1906.
41. Henschen, F.: Anat. Hefte, 26, 575, 1904.
42. Ribadeau-Dumas, L.: Compt. Rend. Soc. Biol., Paris, 54, 484, 1902.
43. Mayer, A., and Rathery, F.: J. d. PAnat. et de Phys., 45, 321, 1909.
44. Prenant, A.. Compt. Rend. Soc. Biol., Paris, 59, 218, 1905.
45. Policard, J.: “Rev. générale d’histolog.,” p. 431, Masson, 1908.
46. Levi, G.: Arch. ttal. di Anat. e di Embr., 10, 168, 1911.
47. Ciaccio, C.: Centralbl. f. allg. Path., 55, 131, 1913.

EXPLANATION OF PLATES 123

EXPLANATION OF PLATES

Technical Remarks: All photographs were taken with a Zeiss micro-
scope on Cramer isochromatic plates. For illumination a 4-5 amp. Leitz
arc-light filtered with a single potassium bichromate (Orth) screen was
used. The ocular used for all pictures is No. 4, the objective for “low
power" is No. 3, for “high power’ No. 6, and for “oil-immersion” No.
1/12 apochromatic. The difference in size of corresponding structures in
different pictures photographed with the same microscopical magnification
is due to slight differences in the length of the bellows of the camera,
which has to be varied according to various circumstances. For printing
Azo “E and F hard” were used.

All preparations reproduced here were fixed in either Regaud’s or
Bensley’s formalin bichromate mixtures, and stained with a modified
“Altmann anilin fuchsin,” in which the destaining of the plasma with
picric acid had been carried over the usual point. This assures a high
contrast for mitochondria. Only Figure 2 is from a copper-chrome-hema-
toxylin preparation.

Figure 1, low power, urea fed just before the death. The proximal
convoluted tubules stand out by virtue of the greater density of the mito-
chondrial content. This is the typical low power picture.

Figure 2, low power, urea fed just before the death, and Figure 3,
low power, urea fed over a long period. In contradistinction to Figure 1,
Figures 2 and 3 show a distinct localized staining reaction of certain
tubular groups, while the neighboring and otherwise same structures re-
main faint in outline. In the left-hand corner of Figure 3 the usual pic-
ture is approached again. The localized staining reaction is due to the
greater mitochondrial concentration in this region and occurs in any
kidney under ordinary circumstances. Figure 3 is somewhat obscured
by faulty illumination.

Figure 4, high power, urea fed just before death, and Figure 5, high
power, no urea fed, represent the usual dogmatic picture of the mito-
chondrial distribution in the kidney of the white rat under ordinary
circumstances. That one dose of urea has no influence on the mito-
chondrial apparatus is seen in Figure 4 by the unchanged appearance and
distribution of the granules. The proximal convoluted tubules stand out
very distinctly. The lumen is narrow. The brush border cannot be dis-
tinguished, for reasons explained elsewhere. The distal convoluted
tubules, connecting segments (Schaltsttick) and the limbs of Henle’s loop
are distinguished easily. The descending limb of the latter is free from
mitochondria. The distal convoluted tubules (segment intermédiaire,
Schweigger-Seidel’s Zwischensttick) are recognized by the wider lumen

124 A CYTOLOGICAL STUDY OF THE KIDNEY CELL

and the fewer mitochondria. A. Glomerulus. B. Proximal convoluted
tubule. C. Distal convoluted tubule. D. Descending limb of H. L.
E. Ascending limb of H. L. F. Connecting segments.

Figure 6, high power, urea fed during entire length of life, and Figure
7, oil immersion, no urea fed at all, show the end stage in granular disso-
lution. There is no difference in size, appearance and arrangement of the
granules. The lumina of the tubules (proximal) are narrow and the
cells are high in either urea-fed and regular-diet animal. In the latter the
chainlike arrangement of the granules is still preserved. This configura-
tion has been described as the end stage of secretion.

Figure 8, high power, urea’ fed just before death, and Figure 9, oil
immersion, urea fed during the entire life, apparently represent distinctive
and different types of granular dissolution. But comparing Figure &
with Figure 12, it is at once apparent that these differences are only indi-
vidual variations of granular dissolution in the same kidney. Both these
pictures are from the same kidney but from different levels. In Figure 8
a clumping of mitochondria into larger bodies staining like mitochondria
is distinctly seen. In Figure 8a this has been brought out still more
markedly by lengthening the bellows of the camera.

Figure 10, oil immersion, urea fed for fourteen days, shows the usual
variations in the height of the cells and the: difference in the mitochondrial
density within one tubule. There is an early granular dissolution present,
but the rodlike arrangement is still preserved; the picture is not different
from the normal.

Figure 11, high power, urea fed during the entire length of life, shows
the typical batonné arrangement described for the normal and which is
not disturbed by intense urea feeding.

Figure 12, high power, urea fed just before death, shows the early
granular dissolution which occurs normally. The brush border is just
faintly visible, not changed and contains a few fine granules with a mito-
chondrial staining reaction.

Figure 13, oil immersion, urea fed during entire lifetime, and Figure
1f, oil immersion, no urea fed at all, show primarily that the collecting
segments of the tubular apparatus is partially free from mitochondria and
that the few mitochondria which are preserit are not altered in any way or
increased in number by prolonged urea feeding.

Figure 15, oil immersion, prolonged urea feeding, and Figure 16, oil
immersion, no urea feeding, show that the glomerular apparatus is not
influenced by a severe strain such as is produced by prolonged urea
feeding.

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