OBSERVATIONS UPON THE MICROANATOMY
OF THE fl‘VIVO QUICK-FROZEN AND DRIED KIDNEY
IN NORMAL AND EXPERIMENTAL CONDITIONS

Thesis for tho Dogma of Pk. D.
MICHIGAN STATE UNIVERSITY
William Charles Waggoner
I963

 

 

 

1:,

This is to certify that the

thesis entitled

Observations Upon The Microanatomy
Of The I_[_t_t_ Vivo Quick-Frozen And Dried Kidney
In Normal And Experimental Conditions

presented by

William Charles Waggoner

has been accepted towards fulfillment
of the requirements for

m—degree inmgy 5‘
Pharmacology

1/2” 19. Cara; ,9

Major profess

Date—Mg! 9. 1963

0-169

  

  
      

i" LIBRARY

Michigan State
University

ABSTRACT

OBSERVATIONS UPON THE MICROANATOMY OF THE
IN VIVO QUICK-FROZEN AND DRIED KIDNEY
IN NORMAL AND EXPERIMENTAL CONDITIONS

by William Charles Waggoner

Conventional histologic slides do not adequately portray
living functional cellular anatomy of kidneys. Previous
attempts to show living morphological details covered only
normal conditions in three species. The present study was
undertaken to expand earlier work and it is an attempt to
visualize kidney anatomy more accurately than heretofore in
normal and experimental conditions.

Kidneys of ten species were frozen, in 2139, cut, lyo-
philized, stained and compared to contralateral kidneys

processed by conventional histologic methods. ;_ vivo

 

frozen-dried kidney sections averaged 34 percent inter-
tubular area and 13 percent tubular luminal area and showed
red blood cell rich blood vessels. Blood stasis, as
indicated by red blood cell packing in capillaries, and
proximal tubular luminal stenosis occurred when intrarenal
pressure relations were altered with blood vessel, ureter,

or lymph duct ligation. Also, lymph duct ligation increased

William Charles Waggoner

intertubular spaces by 45 percent. Renal capsule ablation

removed kidney distension limitations, increased tubular

luminal area, and caused tubular epithelium to stretch and

become thin. Adrenalin, histamine, and glucose injections
altered intrarenal pressure relations and changed glomerular
filtration rates as shown by proximal tubular luminal clo—
sure or dilation. Hypertrophied kidneys possessed signifi—
cantly enlarged tubular, glomerular, and capsular diameters,
but no distinction was made between simple hypertrophy and
hyperplasia. Renal hypertensive kidneys developed tubular
luminal stenosis, decreased intracapsular space (Bowman's
capsule), and intertubular space, and blood stasis in vary—

ing degrees, depending upon the method used for producing

the hypertension. Four day adrenoprival animal kidney

sections showed decreases in intertubular and proximal tubu-

lar luminal space. Draining post mortem kidneys from the

severed hilar vessels before freezing produced ischemic
sections with autolysis and decreased tubular luminal space.
Sections from kidneys, which were drained, frozen, cut,

thawed, dried, and then stained, appeared to be similar to

conventional histologic preparations.

It is concluded that there are vast cellular differences

between kidneys processed by conventional histologic methods

William Charles waggoner

and the ig_vivo freeze-dry technique. Kidney parts appear

quite elastic and renal overdistension is prevented, in part,

by the renal capsule. Large tubular and intertubular spaces

of frozen controls indicate large volumes of extracellular
fluid. The observed interdigitating tubular cells would in-
crease intercellular area and lend greater constructive

support to the functioning organ. Ligation of renal lymph
ducts, renal vein, or ureter may increase intrarenal pressure
significantly to collapse smaller renal blood vessels and, in

turn, increase resistance to renal blood flow. Intravenous

adrenalin appears to have a two—fold effect on renal tubules:

primary tubular closure and secondary tubular dilation. gIntra-

venous histamine drops systemic blood pressure and causes

tubule luminal obliteration. The tubules of a hypertrophy-

ing encapsulated kidney appear to merge into the interstitial

area and encroach on smaller blood vessels, thus increasing

resistance to blood flow. Decreased intertubular and tubular

luminal space in four-day adrenoprival rats likely result

from decreased plasma volume and blood flow.

OBSERVATIONS UPON THE MICROANATOMY

OF THE lg VIVO QUICK-FROZEN AND DRIED KIDNEY

 

IN NORMAL AND EXPERIMENTAL CONDITIONS

BY

William Charles Waggoner

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Physiology and Pharmacology

1963

Introduction .
Historical Survey

Procedure and Materials

Results .

TABLE OF CONTENTS

Discussion and Conclusions

Summary .

References

ii

Page

23

35

74

86

88

LIST OF FIGURES

 

 

 

Figure Page
1. Freezing with Cooled Isopentane . . . . . . . . 26
2. External View of Cryostat-Microtome . . . . . . 27
3. Internal View of Cryostat-Microtome . . . . . . 28
4. Blood Pressure Recording Apparatus . . . . . . 32
5. Rat Blood Pressure Box . . . . . . . . . . . . 33
6. Blood Pressure Records . . . . . . . . . . . . 34
7. Fish Kidney, Conventional Histologic Technique. 37
8. Fish Kidney, ;__Vivo Freeze-Dry Technique . . . 37
9. Frog Kidney, Conventional Histologic Technique. 38

10. Frog Kidney, in ViVO Freeze-Dry Technique . . . 38
ll. Turtle Kidney, Conventional Histologic

Technique . . . . . . . . . . . . . . . . . . . 39
12. Turtle Kidney, ;_ Vivo Freeze—Dry Technique . . 39
13. Pigeon Kidney, Conventional Histologic

Technique . . . . . . . - . . . . . . . . 4O
14. Pigeon Kidney, ;n_yiy9_Freeze-Dry Technique . . 4O
15. Mouse Kidney, Conventional Histologic

Technique . . . . . . . . . . . . . . . . . . . 41
16. Mouse Kidney, In yiyg.Freeze-Dry Technique . 41
17. Rat Kidney, Conventional Histologic Technique . 42
18. Rat Kidney, ;n_yin_Freeze—Dry Technique . . . 42
19. Guinea Pig Kidney, Conventional Histologic 43

Technique . . . . . . . . . . . . . .

iii

Figure

20.

21.

22.
23.
24.
25.
26.
27.
28.
29.
30.

31.

32.
33.
34.
35.
36.
37.

38.

39.

Guinea Pig Kidney, I Vivo Freeze—Dry
Technique .

 

Rabbit Kidney, Conventional Histologic
Technique .

Rabbit Kidney, I Vivo Freeze-Dry Technique

 

 

 

Cat Kidney, Conventional Histologic Technique
Cat Kidney, In Vivo Freeze-Dry Technique

Dog Kidney, Conventional Histologic Technique
Dog Kidney, ;Q_Vivo Freeze—Dry Technique

Dog Kidney, Interdigitating Cell Membranes
Rat Kidney, Renal Artery Ligation . . . . . .
Rat Kidney, Renal Vein Ligation . . . . . .
Rat Kidney, 4 Minute Ureter Ligation .

Dog Kidney, 30 Minute Renal Lymph Duct
Ligation . . . . . . . . . . . . . . . . .
Cat Kidney, Renal Capsule Ablation . . . . .
Rat Kidney, Adrenalin Effect . .

Rat Kidney, Histamine Effect . . . .

Rat Kidney, Hypertonic Glucose Effect . . . .
Rat Kidney, 21 Day Hypertrophy . .

Rat Kidney, Goldblatt Hypertension .

Rat Kidney, Collodion and Gauze Cast and
Contralateral Nephrectomy . . . . . . . . . .

Rat Kidney, Collodion and Gauze wrap of
Hypertrophied Kidney . . . . . . . . .

iv

Page

43

44
44
45
45
46
46
47
48
49

50

51

52

53

54

55

56

57

58

59

Figure Page

40. Rat Kidney, Collodion and Gauze Cast and

Contralateral Nephrectomy after 7 Days . . 60
41. Rat Kidney, Bilateral Adrenalectomy . . . . . 61
42. Rat Kidney, Histochemical Preparation . . . . . 62
43. Effect of Experimental Treatment of Rat

Kidneys on Bowman's Capsule Diameters . . . . . 69
44. Effect of Experimental Treatment of Rat

Kidneys on Glomerular Diameters . . . . . . . . 7O
45. Effect of Experimental Treatment of Rat

Kidneys on Proximal Tubule Inside Diameters 71
46. Effect of Lymph Vessel Ligation in Dog

Kidneys upon Bowman's Capsule, Glomerular, and

Proximal Tubular Luminal Diameters . . . . 72
47. Effect of Renal Decapsulation in Cat Kidneys

upon Bowman's Capsule, Glomerular, and

73

Proximal Tubular Luminal Diameters . . . . . .

LIST OF TABLES

Page

Animals and Anesthetics . . . . . . . . . . . 24
Number of Tubular Cross-Sections and
Percentile Areas of Renal Cortical Spaces . . 63
Symbols . . . . . . . . . . . . . . . . 63
Nephron Diameters in Microns from
Conventional Kidney Sections . . . . . . . . 64
Nephron Diameters in Microns of IQ Vivo

. . . . . . 66

Frozen-Dried Kidneys . . . . . .

Vi

ACKNOWLEDGEMENTS

I wish to express my sincere appreciation to Dr. W. D.

Collings, my major professor, and professor of the Depart—

ment of Physiology, Michigan State University, for his

dedicated, earnest teaching, whereby he unknowingly intro-

duced to me subject matter of two physiological fields

that would later be used in theses projects, and for find-

ing time in a busy schedule to assist and counsel throughout

the project; Dr. R. A. Fennell, professor of the Department

of Zoology, Michigan State University, who kindly loaned

his laboratory and counsel; Dr. F. E. McCoy, pathologist

of the Gratiot Community Hbspital, who patiently instructed

me in my first pathology; Dr. P. G. Crook, professor and

chairman of the Department of Biology, Hope College, who
intellectually goads one to higher levels of biological

learning; the faculty, staff, and employees of the Depart—

ment of Physiology, Michigan State University, and, in

particular, Dr. B. V. Alfredson, professor and chairman;

Dr. and Mrs. R. L. waggoner, my father and mother, whose
interest and financial aid made it possible; and to my wife,

Joanna, for her patience and secretarial assistance during

the course of the project.

vii

This volume is respectfully dedicated

to my wife, Joanna

viii

"There is no difference between structure and function;
they are the two sides of the same coin. If structure does
not tell us anything about function, it means we have not

looked at it correctly."
A. Szent-Gyérgi, 1951

"We must now become literate enough to read function
out of design, for anatomy is the theater in which physi-

ology takes place."
C. Sherrington, 1950

ix

INTRODUCTION

Histology, the study of normal tissues, may be enter-
ing a new era. With the modern, rapid advancement in
technology, new methods of preparing animal tissues have
emerged. The biologist must revise generally accepted
thinking with respect to normal cellular construction of
tissues. What we are accustomed to seeing is not neces-
sarily correct, and adjustments and reconstruction of con-
cepts may be necessary.

It was sometimes tempting from physiological data to
assume various aspects of living organ architecture which
could not be directly observed. However, showing the
correct in_yiyg_construction of many organs has not been
done generally.

An organ of controversy with regard to natural morph-
ological structure is the kidney. Ordinary paraffin sec—
tions do not adequately project the physiological picture.
In this frame of reference, the present study was under—
taken to improve and expand the earlier attempts to por-
tray the probable living kidney microanatomy.

The primary intent of the present work is to show, in
various animals, the functional cellular architecture of

the kidney during normal and experimental conditions.

HI STORICAL SURVEY

Optics

Optical properties of curved surfaces were first de—
scribed by Euclid (590 B.C.), Ptolemy (127—151 A.D.), and
by Alhazan at the beginning of the 11th century. It is
not surprising that spectacle-making and practical optical
science emerged simultaneously. In 1590 the Janssen
brothers combined two convex lenses with a tube and built
the first compound microscope. Later, Leeuwenhoek improved
lenses sufficiently for use in scientific investigation.
Faber of Bamberg, a resident physician in service of Pope
Urban VII, gave the name of "microscope" to this optical
instrument.

In the 17th century Athanasius Kirchner was the first
to employ the microscope systematically in the study of
diseases, although he erred in many of his interpretations.
Later, Robert Hooke modified the optical instrument of
Kirchner with a sub-stage condenser. (Cited by DeRObertis,

s; 11... 1948.)

The Cell

In the 17th century Robert Hocke used his penknife to

shave thin slices of cork and examine them under the

microscope. He observed large pores in the material and
called them "cells," a term used earlier by Roger Bacon.
The cell theory, as an organized unit, actually originated
with Schleiden and was later confirmed by Schwann. Later
R. Virchow in Vienna applied this theory to pathology
demonstrating that pathological processes take place in
cells and tissues. (Cited by DeRobertis, _£_§1., 1948.)
Max Verworn (1895) emphasized the role of the cell in
physiology when he stated: ”It is to the cell that the
study of every bodily function sooner or later drives us.
In the muscle cell lies the problem of the heart-beat and
that of muscular contraction; in the gland-cell reside the
causes of secretion; in the epithelial cell, in the white
blood cell, lies the problem of the absorption of food,
and the secrets of the mind are hidden in the ganglion
cell. . . If then physiology is not to rest content with
the mere extension of our knowledge regarding gross activ-
ities of the human body, if it would seek a real explana-
tion of the fundamental phenomena of life, it can only

attain its end through the study of cell—physiology."

Freeze—Drying
According to Mann (1902) preparation of tissues for

microscopical research by drying is the oldest of all methods.

He relates that Leeuwenhoek prepared muscle samples by
this method in 1720, carried them in his pocket, and cut
sections from time to time for miscroscopic examination.
The procedure used by Altmann (1890) was more refined
than this and involved the maintenance of small portions
of tissue ig_y§gg9_at -20°C., over sulfuric acid, in a
desiccator. After several days tissues were embedded in
ygggg in paraffin, wax. Mann himself made use of Altmann's
method, employing a mixture of solid CO2 and alcohol to
freeze whole animals; subsequently he broke off small
pieces and dried them ig.yagpg_at -300C.. In 1915 Bayliss
used a similar method, but dried his tissues at a slightly
lower temperature. The method was of little practical use
until Gersh (1932) produced the forerunner of all modern
freeze—drying apparatus, and his lead was followed by a
large number of investigators. Modifications of the orig—
inal Gersh apparatus, or of the technique for using it,
have been described by Scott (1933), Goodspeed and Uber
(1934), Hoerr (1936), Scott and Williams (1936), Packer
and Scott (1942), Gersh (1948), wang and Grossman (1949),
Mendelow and Hamilton (1950), Stowell (1951), Glick and
Malstrom (1952), Treffenberg (1953), Jensen (1954), Arcadi

and Tessar (1954), Moberger, Lindstram and Andersson (1954),

Lacy and Blundell (1955), Burstone (1956), Caprino (1956),
and Naidoo and Pratt (1956). The earlier types of appa-
ratus and the theory and practice of freeze—drying have been
described by Neumann (1952). An account of the freeze-
drying technique in biological research has been given by
Bell (1955), and Cabrini (1955) has reviewed the fundamental
process principles. The physicalchemical protoplasm freez-
ing and drying properties have been studied by Kulenkampff
(1956), by Mamulina and Orlova (1955), and also by Hartlieb
(1954).

Pearse (1961) states that the ability to obtain rapid
tissue freezing rate depends on the thermal conductivity of
the liquid used for the freezing bath and, if the pieces of
tissue are large, it will also depend substantially on their
own conductivity. Scott (1933), HOerr (1936) and Simpson'
(1941a and b) have stressed the necessity for using a cool-
ing liquid of high conductivity at a temperature as low as
possible. In his earlier experiments Gersh used liquid air
(-l95°C.) for his quenching bath and, although this had a
sufficiently low temperature, its conductivity was low
because a vaporized air layer around the specimen prevented

sufficiently rapid transference of heat.

In order to overcome this effect Scott (1933) recom-
mended the use of ethyl alcohol cooled to -100°C. in place
of liquid air, but this method had the disadvantage that
ethanol is solid at —115°C. and very viscous at higher
temperatures in this region. Hoerr (1936) introduced the
method of placing the tissues in crude isopentane cooled
to about —165°C. with liquid nitrogen. Crude isopentane
solidifies at —190°C. and the temperature at which it is
beginning to become viscous is usually employed. Hoerr
also used isopentane cooled to -120°C. Various other
fluids have been employed for cooling. Emmel (1946) tried
isopentane mixed with dry butane to lower the melting point
of the former, and BelI (1952) proposed a mixture of pro-
pane (3 volumes) and isopentane (1 volume) or, alternatively,
pure propane, which has a melting point at -l85°C. Some
authors, surprisingly,continued to use liquid air directly
for quenching (Pease and Baker, 1949) and found no advan-
tage in using isopentane. Simpson (1941a) confirmed the
excellence of isopentane technique but maintained that
even by this means the resultant freezing was uneven unless

very small pieces of tissue were used. He described three

zones in the tissue caused by quenching: an outer zone in

 

which ice crystal formation was minimal and preservation
of structure excellent, an intermediate zone in which
structural distortion by large crystals was particularly
bad, and an inner zone of better but relatively indif-
ferent preservation. Simpson believed that structural
changes in the intermediate zone were caused, while it was
still not solid, by pressure from the hard frozen outer
zone. Bell (1952), on the other hand, considered it re-
presented a zone in which solidification temperature was
too high for rapid nucleation to occur, but crystal growth
rate was high. In the innermost zone, according to this
author, solidification temperature is too high for rapid
nucleation but also too high for crystal growth rate to
produce large artifacts of the intermediate zone.
According to Simpson (1941b) the best practice for
tissues required for cytological examination is to regu-
late the size of the sample and the temperature of the
quenching bath so as to obtain entirely outer zone preser—
vation. No means exist for calculating these factors for
different tissues and the method of trial and error must
be employed. Bell (1952) conducted a series of experiments
regarding the speed of quenching. By placing a thermo-

couple within a length of excised mouse intestine and

plunging the tissue into cooled isopentane, he found the
time taken to reach equilibrium with the bath averaged 10
seconds. The blank for the thermocouple was 2 seconds.
The critical temperature of water (-40°C.) was reached in
4 seconds. Parker, gt_a1. (1962) did a similar set of ex-
periments in mouse kidneys. With the tip of the thermo-
couple 1 mm. below the surface of the kidney, the tempera-
ture was found to fall from 37°C. to -lOOC. in less than
0.1 sec. and to -1000C. in 4 sec. With the thermocouple
placed in the middle of the kidney, some 3 mm. from the
surface, the temperature drop was slower: to -10°C. in

4 sec. and to -1oo°c. in 8 sec.

Crvostat-Microtome
Freezing microtomes provided with means for cooling

the knife blade with an additional CO jet were introduced

2
into histological technique by Schultz-Brauns (1931a and b,
1932). Commercial models have been available for several
years and have been used by a number of workers (wachstein
and Meisel, 1953) to obtain semi-serial sections for histo-
logical use.

The first cryostat—microtome was developed in Denmark

by Linderstrom-Lang and Morgensen (1938) for their studies

in quantitive histochemistry in which alternate frozen sec-
tions were taken for chemical estimation and histology. In
the original model cold was maintained by blocks of dry ice,
and from this to provision of refrigerating coils was a sim-
ple step. The microtome was modified with a glass plate in
front of the knife edge which effectively prevented section
rolling or curling. No real attempt was made to apply the
cold microtome technique thus developed by the Danish authors
to histological methods in general. In 1951 Coons and his
collaborators made a number of technical improvements in
designing a new cryostat-microtome of 6 cubic feet capacity

for working temperatures between —160C. and -18°C.

Intrarenal Pressure Relationships

Brodie (1914) and Brodie and Mackenzie (1914) quick-fixed
kidneys of normal and experimental animals in formalin and
measured nephron diameters. Using Poiseuille's equation and
tubule measurements of control dog kidney paraffin sections,
he calculated the glomerular driving pressure required to pro-
pel filtered urine the nephron length as 72 mm. Hg for an es-
timated flow of 1 ml. per minute. On histological slides of
cat kidneys following decapsulation he found a profound in-

crease in intracapsular volume of Bowman's capsule and an

10

increase in tubule diameters, although the increase of the
lumen diameter was greater than outside tubule diameter. In
kidneys with ligated ureters, blood stasis was present in the
capillary beds and tubular luminal diameters were increased
slightly.

Brodie reasoned that the tough kidney capsule prevented
overdistension and he observed that after decapsulation the
kidney weight increased over controls by 20%. Renal vein
ligation, with decapsulation, produced no greater weight in—
crease than decapsulation alone. Ureter ligation increased
kidney weight after 5 to 15 minutes. He also observed that
a diuretic kidney had a 30% gain in weight over a "resting"
kidney.

Generally, Brodie concluded most kidney parts are highly
elastic and overdistension is prevented by general inexten-
sibility of the connective tissue framework and, most of all,
the capsule; and glomerular pressure is transmitted through-
out the tubules, transversely and longitudinally.

In 1950 Montgomery §£_§1, blindly inserted blunt hypo»
dermic needles directly into the kidney cortex and measured
pressures of 20—25 mm. Hg. Later, Gottschalk (1952) used a

100 micron pipette in the same fashion and measured pres—

sures of 4 to 19 mm. Hg in 65 rats, guinea pigs, rabbits,

11

and cats, and 12 to 25 mm. Hg in 8 dogs. They both believed
that they were measuring intrarenal interstitial pressure.
Swann §£__1, (1955, 1956a & b, 1958, and 1960) showed
that fluid draining from kidneys after excision was differ-
ent from blood. The hematocrit was one-half normal systemic
values. Most of the fluid drained from the renal vein and
very little from the artery or ureter. They believed drain—
ing blood had been diluted by renal interstitial fluid. With
a series of formulas, he calculated what he believed was the
interstitial volume of the living distended kidney. He con-

sidered interstitial fluid to be a mixture of systemic blood

t al. (1957) used

plasma and tubular resorbate. Overbaugh
Swann's (1955) procedure and found species variation inter-
stitial volume: (expressed as percent of total functional
kidney weight) 18.6% in mice, 23.0% in rats, 20.7% in cats,

and 32.2%.in dogs. Swann §§_§1, (1958) showed that distend-
ing interstitial fluid contained much protein, and concluded
that it migrates easily from the vascular bed to the inter-
stitial space and back. Pease (1955a), in electron microscope
studies, showed that the kidney vascular bed is very porous
at the capillary endothelium with holes 400-500 Angstroms
wide. Along this same line, but With a different experimen-

tal approach, Collings and Swann (1958) infused dog kidneys

12

with radioactive blood albumin and hypothesized penetration
of albumin into the interstitial compartment.

Gottschalk (1952) ligated renal arteries of rats and
showed that, although renal artery blood pressure dropped
markedly and urine flow stopped, interstitial pressure
was maintained at control levels. He consistently found
interstitial pressure greater than venous pressure, and when
the renal vein is ligated for a period of time, interstitial
pressure and venous pressure become equal. After ligating
ureters, interstitial pressure increased and, in addition
noted that ureteral ligation in rabbits produced a swollen
and cyanotic kidney resembling an organ congested by in-
creased venous pressure. He reasoned that renal venous
blood pressure contributes, in part, to the values of tubu-
lar and interstitial pressures.

Winton (1937) found a drop in urine flow during a rise
in venous pressure or a drop in arterial pressure. Later,
Winton (1951), Hinshaw _t_§1, (1959), and Hinshaw _t_§l.
(1961) found a decrease in renal blood flow with ureter
ligation.

Using the studies of Sugarman gt 31. (1942), LeBrie and

Mayerson (1960) found that lymph flow in the dog kidney

13

increased about five times during partial occlusion of the

t al, (1958) also showed an increase in

vena cava. Haddy
lymph flow with an increase in venous pressure.

As shown by Winton (1956), Gottschalk (1952), and
Gottschalk and Mylle (1956), renal tissue pressures may
rise to high levels subsequent to an increase in tissue
fluid, vascular volume, or tubular volume. Haddy gg_§l.
(1958) interprets this as a result of indistensibility of
both the kidney stroma and renal capsule. They reason,
therefore, that elevating venous pressure may increase
renal vascular resistance, in part, because greater filling
of veins raises tissue pressure, thus partially collapsing
high resistance vessels.

In micropuncture studies in rats, Gottschalk and Mylle
(1956) found a mean intratubular pressure of 13.5 mm. Hg.
After uretral and renal vein ligation they found an increase
in intratubular pressure. They also noted, occasionally,
an abrupt obliteration of tubular lumina while inserting
mircopipettes into the tubules of functioning rat kidneys.

They report: Collapse* of all or a portion of the visible

 

* In the sense of disappearance of the lumen (Gottschalk,
1962)

l4

tubules sometimes occured after accidental trauma to the
animal, i.e. hemorrhage or anoxia, and rarely without
demonstrable cause. Collapsed tubules appeared white and
opaque, and their lumina were more or less obliterated."

In 1930, Edwards examined, under magnification, lg
yiyg_ frog kidney tubules and their means of handling dyes.
He observed that when a glomerulus had little or no blood
flow through it, the dye in the tubule became very concen—
trated. This dye was apparently stationary in the tubule.
He says: " . . . a coagulable or precipitable exudate
appears in them." Later, when he made these same kidneys
diuretic, the glomeruli became active and the tubules
dilated. Edwards states: "That this dilation is the
result of fluid flow through the tubule is shown by ob-
serving the concentrated column of dye become separated
from the tubule walls on all sides, and as the relatively
clear fluid rushes past this static column of dye and the
tubule wall becomes tinged, cuff like, around the dye,
while the fluid next to the wall remains untinged. Finally,
the dye is washed entirely from the lumen."

With excellent electron microscopy Pease (1955a & b)
shows extraordinary folding or intussusception, with var-

iations in tubular plasma membrane spacing, demonstrable

15

in the basal region of tubular cells, and shows tubular
basement membrane surface area to the interstitium may
be vastly increased. He has no doubts that the brush
border represents a simple extension of apical cytoplasm.
Further, he states: "The apical portion of the proximal
tubule is very sensitive to osmotic variables and appears
radically different after seemingly minor changes in fix-
ation procedure. Terminal changes indicate that the
apical end of these cells is very labile. This portion
of the cell may thus be swollen to such an extent that
the tubular lumina will be obliterated and the brush pro-
cesses jammed together." With the electron microscope
Herman and Hanssen (1962) did similar studies and showed
the apical border of the proximal tubule is quite sensi-
tive to post mortem changes.

Parker §£_§1, (1962) fixed mouse kidneys in yiyg_by
ultra-quick freezing. The frozen kidneys were sectioned
on a cold microtome, dehydrated, and stained for micro-
scopic examination. All tubular lumina were found to be
widely open, with tubular walls thin and stretched out;
and a large intertubular space was observed. They also
reported that if fixation by freezing is delayed for 10-

20 seconds, the tubules have become filled with an

16

amorphous eosinophilic staining substance and the lumen
reduced to a mere slit or obliterated. Hanssen (1960)
and Herman and Hanssen (1962) showed similar findings,
and, furthermore, found that luminal obliteration can
be prevented by making animals diuretic.

By cutting with scissors, photographs of i__vivo fro-

 

zen kidney slides and then weighing the respective parts,
Parker §;_§1, (1962) found sections to consist of 45%
tubular cells, 15% lumina, and 43%.intertubular space,
excluding all glomeruli and large blood vessels from the
measurement. Peritubular capillaries were included in
intertubular space measurement. Babin §§_§1. (1961)
attempted to show post mortem maps (relative areas) in
renal disease studies and found an increase in inter-
stitial area with hypertension in contrast to Swann g§_al.
(1959), who believe that kidney interstitial spaces dimin—

ish in hypertension.

Drugs

“In an experiment that has long been considered a
classic in renal physiology, Richards and Plant (1922a & b)
showed that when a rabbit kidney is perfused at a pressure

such as to maintain a constant blood flow, adrenalin causes

17

a rise in the requisite perfusion pressure and, at the same
time, an increase in kidney volume, an observation confirmed
in eviscerated rabbits and dogs. A rise in perfusion pres-
sure can only mean increased renal resistance, presumably
because of vasoconstriction, while an increase in renal vol-
ume points to vasodilation. Richards and Plant interpreted
this paradox by suggesting that adrenalin acts preferentially
to constrict the efferent glomerular arterioles, thus causing
enlargement of the glomeruli and the preglomerular vessels
and swelling of the kidney. This phenomonen has often been
confirmed by Smith (1939~40), and it has been widely accepted
that preferential efferent arteriolar constriction, by
raising the glomerular pressure, would maintain the filtra-
tion rate despite a reduction in flood flow." (Cited by
Smith, 1951.)

Ahlquist _t_§1, (1954) injected epinephrine aS'a single
rapid intravenous slug and noted that the renal blood flow
decreased acutely. The constriction, presumably at the af-
ferent glomerular arterioles, quickly disappeared leaving
the renal blood flow subject to the elevated arterial pres-
sure. The flow changes were reflected in the urinary out-
put. During the acute constrictor phase, the urinary output

ceased. When the flow increased, due to the elevated

l8

pressure, the urine output was increased above control
levels. Although Smith (1951) presumed the increased
kidney volume, accompanying adrenalin infusion, was due

to the interstital compartment expansion, Swann 33 al.
(1952a) found that renal interstitial pressure drops with
pressor doses of adrenalin. Using ip_yiyg dyes and histo-
kxnc sections, Winton (1937) injected adrenalin, intra—
venously, and observed that the number of active renal
glomeruli was decreased, however, Smith (1951) thought
that the reduction in renal plasma flow induced by adre—
alin is not accompanied by closure of any glomeruli, as
was shown by the glucose tubular reabsorption, which re-
mained at constant maximum rates. Page and McCubbin (1953)
also considered adrenalin a renal vasoconstrictor, while
Goodman and Gilman (1958) state adrenalin may reduce renal
blood flow by 50%.

In human patients Bjering (1937) observed that hista-
mine reduced both the creatinine and urea clearances, but
simultaneous reduction in mean arterial pressures made
the renal effects difficult to interpret. In addition,

Page and McCubbin (1953) considered histamine a renal

vasodilator.

19

In histologic sections of duretic dog kidneys, which
were quick-fixed in formalin, Brodie (1914) observed an
increase in glomerular and Bowman's capsule diameters.
Gottschalk and Mylle (1956) found that rat kidney intra-
tubular pressures increased with intravenous hypertonic
glucose injections. Bloom and Fawcett (1962), in histo—
logic sections of formalin—fixed diuretic kidneys, found
kidney tubular epithelium flattened with an extended
brush border. Longley and Burstone (1962) believed that
postmortem swelling of proximal tubules has an osmotic
basis, as it is reduced or eliminated in kidneys perfused

and fixed in hypertonic solutions.

Renal Hypertension

Hypertension syndrome is described back as far as
2600 B.C. in the Yellow Emperor's Classic of Internal
Medicine as the illness associated with a hard pulse.
In 1694 Giorgio Baglivi of Rome performed a necropsy
on the body of the famous Italian physiologist and ana-
tomist, Marcello Malphighi of Bologna, and indicated
Malpighi's fatal apoplexy was due to cerebral hemorrhage
secondary to renal hypertension. Stephen Hale's studies

were by far the most significant contribution of the 18th

20

century toward hypertension research, when he reported
experimental proof that flowing blood exerts a pressure
on the walls of blood vessels. Although induration of
the kidneys with oliguria, hematuria, and edema had been
described by physicians for more than 1,000 years, it
remained for Richard Bright, in 1827, to associate clin-
ical findings of albuminuria, hardness and fullness of
pulse, edema, and hypertrophy of the left ventricle with
pathologic findings of sclerosing, contracted kidneys.
(Cited by Skelton, 1962.)

In 1898 Tigerstedt and Bergman attempted to draw
attention to the possible importance of a blood-pressure-
raising substance formed in the kidney. Ruyter (1925)
first described a peculiar type of kidney cell, which
seemed to be in the afferent arteriole region of the
glomerulus. It was left to Goormaghtigh (1939) to directly
correlate hypergranulation of these juxtaglomerular cells
with a degree of renal ischemia and hypertension.

In 1940 Page and Helmer and BraunauMenéndez §£_§1,
(1946) simultaneously extracted renin from kidneys of
several species and it was regarded as a proteolytic
enzyme which acts on the alpha-Z-globulin of plasma to

produce the pressor polypeptide, now known as angiotensin.

Soskin and Saphir (1932) readily produced renal hyper-
tension by enclosing the kidney(s) in a collodion-gauze
cast. Later, Goldblatt _E_§l, (1934) initiated an avalanche
of hypertension research by placing silver clamps on renal
arteries, reducing renal blood flow, and causing sytemic
hypertension.

With the Goldblatt kidney Mason _§__1, (1940) found
renal arterial pressure dropped distal to the clamp which
was placed on the renal artery. After hypertension ensued
and blood pressure in the kidney returned to control states,
systemic hypertension still remained. They also noted no
decrease in oxygen consumption of experimental kidneys.
Corcoran and Page (1941) noted that kidneys under chronic
Goldblatt constriction showed no decrease in blood flow
and could not, therefore, be correctly designated "ischemic.“

Swann §£_a1, (1952b, 1959) produced perinephric hyper-
tension, after Soskin and Saphir, and found interstitial
pressure values from 25 to 60 mm. Hg. H0wever, the Gold-
blatt kidney showed no change in interstitial pressure.

In the collodion-gauze encapsulated kidney Swann showed
weight gain was as fast as hypertrophying kidneys and fluid
volume draining from the wrapped kidney was greatly de-

creased. Therefore, he reasoned, the renal cells hyper—

trophied into intercellular spaces and decreased

22

pre-existing values of interstitium.

MacKay §£_§1, (1938) and Moustgaard (1948) showed that
hypertrophying kidneys increase in both wet and dry weight.
Using tritiated thymidine and DNA assay, Miyada and Kurnick
(1961) found increased incorporation of the former and in-
creased content of the latter in rat kidneys following un-
ilateral nephrectomy. These studies indicated both hyper-
plasia and hypertrophy play significant roles in compensatory
renal growth. McCoy (1960) got similar results in control
kidneys, but noted no dry weight gain in collodion and
gauze-wrapped kidneys of hypertensive rats. He interpreted
this as a mere hypertrophy with no hyperplasia in a wrapped

kidney during hypertension.

Effect of Adrenalectomy

In 1962 Swann drained post mortem kidneys of rats, 4 days
after total adrenalectomy, and showed the volume of blood
in renal distending fluids to be 4/5 of controls. Also, the
volume of fluid, which had been measured as interstitial
fluid, shrank to 1/5 its normal value. These results were
probably due to a decrease in plasma volume, body weight,

and blood pressure (Turner, 1955).

PROCEDURE AND MATERIALS

Animals
Initially, kidneys of fish, frog, turtle, pigeon,
mouse, rat, guinea pig, rabbit, cat, and dog were studied

comparing tissues of conventional histologic procedures

to those of the in_vivo frozen-dried technique. Secondly,

in vivo frozen—dried kidneys of experimental rats, cats,

 

and dogs were studied. All animals were carefully screened
and examined for disease. Dogs were negative for Lepto-
spira pomona, icterohemorrhagiae, and canicola blood anti-
bodies. In addition, dogs were given Leptospira canicola-
icterohemorrhagiae and canine distemper vaccines (Fromm).

\

At least three animals of each kind were used in all studies.

lg_Vivo, Freezing-Drying Technique

Animals were anesthetized according to Table 1 and placed
in a supine position. A lengthy midline incision opened
fully the peritoneal cavity. The kidneys were exposed free-
ing them from all adherent tissue except for the hilar
vessels and the ureter. Not less than 50 ml. of 2—methy1-
butane (Eastman) were cooled with liquid air to -l75°C. .
Temperature measurements were made with a cryoscopic thermo-

meter (Sargent - S 80135). The cooled isopentane was poured

23

TABLE 1

 

 

ANIMAL

ANESTHETIC

 

1. Fish
(Salmo gairdneri})*

 

2. Frog
(Lana W)

3. Turtle
(Chrysemys picta)

 

4. Pigeon
(Columba livia)

 

 

5. Mouse
(Mus familiaris)

 

6. Rat
(Rattus rattus)
Carworth Farms Nelson

7. Guinea Pig
(Cavia porcellus)

 

8. Rabbit
(Orygtolagus cunigglgs)

9. Cat
(Felis catus)

10. Dog
(Canis familiarig)

 

Tricaine Methanesulphonate
(MS222) (Sandoz) Swim in
1:20,000

Ethyl Carbamate (Merck)
5 mgm./gm. (dorsal lymph sac)

Double Pith

Diethyl Ether
(Merck)

Diethyl Ether

Diethyl Ether - surgery
Sodium Pentobarbital — blood
pressure (Abbott) 30 mgm./Kg.
Intraperitoneal

Diethyl Ether

Diethyl Ether

Diethyl Ether

Sodium Pentobarbital, 30 mgm./Kg.
Intravenous, Surgery (Abbott)

Sodium Pentothal, 20 mgm./Kg.
Intravenous (hypnotic dose)
blood pressure (Abbott)

* Classifications from Spector, 1956.

24

25

in a steady stream on the exposed renal surface. This
freezes one side rapidly, but the side adjacent to the
abdominal wall freezes slowly. Hence, only tissues
from the exposed renal surface were prepared for micro-
scopic study. The frozen kidney was grasped with cold
tongs, cut loose with a cold surgical blade, and plunged
into powdered dry ice.

Tissues were frozen to a chuck and cut at -150C. and
8 microns on a Harris International Cryostat-Microtome
(Model CT). Sections were placed individually on -15°C.
coverglasses, seated in —150C. miniature spacing racks,
and placed in a Vertis lyophilizer (Model 10-102) for 18
hours at 20 microns of mercury. Following lyophilization
tissues were stored in a dessicator until they were
fixed to albuminized slides. Then, slides were flooded
on a staining rack with alcoholic Wright's stain (Hartman-
Leddon) for two minutes. Distilled water was added for
one minute before the slide was washed thoroughly.
Tissues were destained and cleared in ethanol, acetone,
and xylene, respectively. Mounting from xylene was with

Permount (Fisher) and glass coverslips.

26

 

Fig. l. Freezing with Cooled Isopentane.

Isopentane (2—methy1butane) is poured into the large
Pyrex test tube, which is then lowered into the Dewar flask
containing liquid air. The cooling period, lasting about
five minutes, brings the isopentane to -l750C. before it

is poured over the functionally distended kidney.

 

Fig. 2. External View of Cryostat—Microtome.
Small thermostat button on the right side below rotary
crank regulates cabinet temperature. Tissues are handled

through the clear hinged lid. Apparatus is portable.

 

Fig. 3. Internal View of Cryostat-Microtome.

Sections slide off kidney between blade and anti-roll
plate, and are placed on —15°C. coverslips, which are
seated in the small plastic spacing rack (bottom right)
for lyophilization. Present section thickness setting is

at 8 microns (2 microns per division).

29

Conventional Histolggical Technique

Kidneys from freshly euthanized animals were fixed
24 hours in 10% formalin. After processing through the
conventional solvents, tissues were blocked in paraffin,
cut at 4 microns, placed on albuminized slides, and stained
with hematoxylin and eosin. Mounting from xylene was

with Permount and glass coverslips.

Vessel Ligation

In the rat the renal artery or vein was ligated for
30 seconds before kidney freezing. The ureter was ligated
for 4 minutes previous to freezing. To augment lymph
flow a dog was fed a pint of cream. The superior and in-

ferior polar renal lymph ducts were ligated for 30 minutes

prior to kidney freezing.

Renal Capsule Ablation

In the cat the renal artery was clamped and the organ
became flaccid and shrank. The renal capsule was quickly

stripped off and blood flow re-established for 5 minutes

before kidney freezing.

30

Drugs

0.1 cc. Adrenalin chloride (Parke—Davis), 1:50,000,
or 0.1 cc. histamine diphosphate (Abbott), 1:2,000, was
injected via the vena cave in three rats, respectively.
Thirty seconds ensued before kidney freezing. 0.5 cc. of
20% glucose was injected in three rats. Gross ureter

peristalsis was observed before kidney freezing.

Hypertrophy and Renal Hypertension

Hypertrophy was obtained by unilateral nephrectomy and
an ensuing period of not less than 21 days. Renal hyper—
tension was induced by the Goldblatt (1934) method or vari-
ations of Soskin and Saphir (1932). These were: (A) Con-
striction of the left renal artery by a silver clamp and
right renal ablation; (B) Collodion and gauze wrap of the
left kidney and right renal ablation; (C) Collodion and
gauze wrap of a hypertrophied kidney; or (D) Collodion
and gauze wrap of the left kidney and right renal abla-
tion after one week. Hypertension had been at least 7

days in duration at kidney freezing.

Adrenalectomy

Bilateral adrenalectomy was performed in three rats

four days before kidney freezing.

31

Blood Pressure Measurements

Using the principle of Prioli and Winbury (1960) and
Friebel and Vreden (1958) blood pressures were taken with
the Physiograph (E & M) employing a cuff and pulse—sensi—
tive transducer (see figures 4 & 5.). Systolic pressures
were obtained from the d095' hind feet and rats' tails.

As shown in figure 5, a special warming box was used for

rats.

Microphotography,

All tissue photographs were with a Labolux (Leitz)

microscope and Leica (Leitz) camera employing apochro-

matic (Zeiss) objective. Pantatomic-X and Kodachrome II

Professional films (Eastman) recorded the slides.

Histological Slide Measurements

To show area percent occupied by kidney glomeruli,
tubular cells, tubular lumina, and extracellular spaces,
tracing paper was superimposed on kidney photographs and
the respective structures were sketched. The sketches
were cut out and the relative pieces weighed.

Direct diameter measurements were made on histological

slides using a Microstar (A & O) binocular microscope em-

ploying a calibrated micrometer ocular.

32

 

Fig. 4. Blood Pressure Recording Apparatus.

Leads from the pressure cuff and pulse—sensitive trans—
ducer, coming from the rat blood pressure box, plug into the
small electrosphygmograph base (upper left center). This
instrument, integrating pulse wave and blood pressure
recordings, plugs directly into a Physiograph amplifier. No

special warming apparatus is necessary for dogs.

 

 

 

Fig. 5. Rat Blood Pressure Box.

The anesthetized rat (sodium pentobarbital) lies on a
cushion with his tail through the cuff. A pulse transducer
is attached to the tail's ventral side distal to the cuff.
Two 40 watt light bulbs under a reflector (right) warm the
tail and cause vasodilation.

In the anesthetized dog (sodium pentothal) the human
style pressure cuff is wrapped on the hind leg and the pulse

transducer is fitted on the ankle.

34

RAT TAIL PULSE WAVE
"‘"WW“uwuwm WM...“ \I\. pm“

'rl‘rrrrnrxrrrrfrwwvvmwvv“wme/w‘rvwn J‘V‘I‘IJ‘IJ‘J‘WV‘V‘V‘V‘VIV‘ V‘Ifl \‘V‘FV’
|\\‘I\\‘\\\'I\\II|\I\\.\\\\I\\II\I I\\I\\\\\‘Itg‘t.\III\IIIIII|

200
I50

 

Fig. 6. Blood Pressure Records.

The top graph shows an amplified pulse wave from a rat
tail using the pulse transducer and Physiograph. Note that
the wave includes a dicrotic notch. Heart rate was 196/
minute.

On the same animal, the lower graph shows periodic cuff
inflation and pressure decay. The pulse begins to make a
strong appearance at 140 mm. Hg. Similar recordings are

obtained with dog.

RESULTS

As seen in figures 7 through 27, the i§_yiyg freeze-dry
technique reveals microscopic details quite different from
those conventionally pictured by histologists. In control
kidney sections, all tubular lumina are widely open with thin
epithelium. Large intertubular spaces are present containing
red blood cell rich peritubular capillaries. Glomeruli, rich
in red blood cells, project into Bowman's capsule leaving an
intracapsular space. Figure 27 shows interdigitating cells
found in the dog's thicker renal tubule walls. Effects of
various experimental renal treatments are pictured in figures
28 through 42. Bloom and Fawcett (1962), Andrew (1959), and
Edwards and Condorelli (3928) were used as references to
morphological and microscopic detail for the various species
studied.

Table 2 shows the percent of total renal cortical area
or space devoted to glomeruli, tubular cells, lumina, and
intertubular space, along with the number of tubular cross-
sections used in each calculation. In this sort of kidney
“map" study, peritubular capillaries were included as part

of the intertubular compartment.

The symbols shown in Table 3 explain the column headings

35

36

in Tables 4 and 5. Table 4 is a listing of nephron diameters,
in microns, of conventional histologic kidney slides in ten
species. Table 5 is a listing of nephron diameters, in mi-

crons, of in vivo frozen—dried kidneys. For nephron diameter

 

data, at least three structures on each of at least three
different kidney slides were measured. In most cases ten
structures of each classification were examined. Only those
nephrons which were judged to be cross—sectioned at right
angles to the long axis of the nephron were measured.
Glomeruli, which were obviously sectioned acentrically, were
omitted. Statistically, the sample means of the controls
and experimentals were compared using the small sample method
for fewer than 30 observations.

The graphs in figures 43 through 47 summarize the effects
of renal treatments upon Bowman's capsule, glomerular, and

proximal tubular luminal diameters.

 

Fig. 8. Fish, In Vivo Freeze—Dry Technique, Wright's, x300.

 

 

Fig. 9. Frog, Conventional Histologic Technique, H. & E.,
x300.

 

n Vivo Freeze—Dry Technique, Wright‘s, x300.

Fig. 10. Frog,

 

 

Fig. 11. Turtle, Conventional Histologic Technique, H. & E.,
x300.

 

 

Fig. 13. Pigeon, Conventional Histologic Technique, H. & E.,

 

Fig. 14. Pigeon, In Vivo Freeze-Dry Technique, Wright's,
x300.

 

Fig. 15. Mouse, Conventional Histologic Technique, H. & E.,
x300. ‘

 

Fig. 16. Mouse, ;_ Vivo Freeze-Dry Technique, wright's,
x300.

 

 

Fig. 17. Rat, Conventional Histologic Technique, H. & E.,
x300.

 

 

Conventional Histologic Technique,

Guinea Pig,

Fig. 19.

H.&E.

, x300.

 

Wright's,

y Technique,

 

IQ Vivo Freeze-Dr

Guinea Pig,

Fig. 20.
x300.

 

Fig. 21. Rabbit, Conventional Histologic Technique, H. & E.,
x300.

9‘ ‘3‘“ .v.. p
8)..» k . ‘ I ,
95:: .wf‘W3‘QS

”2?» E599-

‘K‘ a
on ' .v. .I

Fig. 22. Rabbit, IQ Vivo Freeze-Dry Technique, wright's,
x300.

 

"I
I? ”a. 'fi'”

 

45

. ”what; .4“ 3m" ' "’a‘
t 6&4 : ““Vfiw . 5;: ' at?"
. . . q. I...“

      
  

  

Fig. 23. Cat, Conventional Histologic Technique, H. & E.,
x300.

~.

~ .3.

 

1"".‘DW'1A

Fig. 24. Cat, In Vivo Freeze—Dry Technique, Wright's, x300.

46

 

H. &E.,

Dog, Conventional Histologic Technique,

Fig. 25.
x300

 

Wright's,

 

In Vivo Freeze—Dry Technique,

Dog ,

Fig. 26.

47

 

Fig. 27. Dog, B.P.* - 120 mm. Hg, No Treatment, IQ Vivo
Freeze-Dry Technique, Proximal Tubule, Wright's, x2,000.

Nuclei appear to be stellate with interdigitating cell
membranes. Mean nuclear diameter is 6.25 microns. Inter-
digitating cellular projections have a mean diameter of 1.4
microns. Canaliculi created by projections have a mean

width of 2 microns.

* Blood pressure

48

 

Fig. 28. Rat, B.P. - 117 mm. Hg, Renal Artery Ligation 15
sec. Before Kidney Freezing, ;p_Vivo Freeze—Dry Technique,
Wright's, x300.

The majority of tubule lumina are stenosed or obliter-
ated with an eosinophilic staining substance. Space in
Bowman's capsule is virtually absent. Several tubules,

which appear to be distal, collecting, and Henle thin seg—

ments, remain open. Intertubular space is still abundant

(see figure 18).

 

49

 

Fig. 29.

Rat, B.P. — 130 mm. Hg, Renal Vein Ligation 30

sec. Before Kidney Freezing, lg Vivo Freeze-Dry Technique,

Wright's,

Most
although
prevails
with red

controls

x300.

of the tubule lumina are stenosed or obliterated,
the thin segments are more distended. Some space
in Bowman's capsule. Capillaries are engorged

blood cells. Intertubular space appears to equal

(see figure 18).

 

Fig. 30. Rat, B.P. — 105 mm. Hg, Ureter Ligation 4 Minutes
Before Kidney Freezing, lg Vivo Freeze—Dry Technique, Wright's,
x300.

All tubules appear to be open and slightly more dis—
tended than controls (compare with figure 18). Capillaries

contain more red cells and the intertubular space appears

to be less than in controls.

 

Fig. 31. Dog, B.P. - 130 mm. Hg, 30 min. After Ligation of
Renal Lymph Ducts, I§_Vivo Freeze—Dry Technique, Wright's,
x300.

This shows an increase in intertubular space. Most
tubules are collapsed and space in Bowman's capsule is

sparse. Compare with figure 26. Quantitatively, inter—

tubular space here is 145% of controls.

52

 

Fig. 32. Cat, Kidney Frozen After Renal Capsule Ablation,
IQ Vivo Freeze-Dry Technique, Wright's, x300.

In comparison with figure 24, the tubules here have a
greater inner and outer diameter with a thinner epithelium.
Diameter of Bowman's capsules appears to be greater with

no apparent change in the glomerular diameter.

 

Fig. 33. Rat, Control B.P. — 95 mm. Hg, 0.1 cc. 1:50,000
Adrenalin Chloride I.V., In Vivo Freeze—Dry Technique,
Wright's, x300.

Several tubule lumina are stenosed with the eosinophilic
staining material. Space in Bowman's capsule is decreased.

Comparing to figure 18, intertubular spaces appear to be

about equal.

54

 

Fig. 34. Rat, Control B.P. - 112 mm. Hg, 0.1 cc. Histamine
Diphosphate I.V., In Vivo Freeze—Dry Technique, Wright's,
x300.

This appears similar to figure 28. The majority of
tubule lumina are stenosed or obliterated with an eosinophilic
staining material. Space in Bowman's capsule is sparse.
Intertubular space is still abundant, although not as great

as figure 28.

55

 

Fig. 35. Rat, Control B.P. — 127 mm. Hg, 0.5 cc. 20% glucose
I.V., In Vivo Freeze-Dry Technique, wright's, x300.

A11 spaces are clearly evident. Bowman's capsule and
glomeruli appear larger than controls (see figure 18) and

tubules have a greater inner and outer diameter.

56

 

Fig. 36. Rat, Control B.P. - 140 mm. Hg, 21 day hypertrophy,
In Vivo Freeze-Dry Technique, Wright's, x300.

Bowman's capsule, glomeruli, and inner and outer tubule

diameters are larger than controls in figure 18.

 

Fig. 37. Rat, Control B.P. - 112 mm. Hg, Silver Clamp on
Renal Artery with Contralateral Nephrectomy, Hypertensive
B.P. - 140 mm. Hg, In Vivo Freeze—Dry Technique at 7 days,
Wright‘s, x300.

There is an apparent increase in Bowman's capsule,
glomerular, and inner and outer tubular diameters. Some
tubular lumina are obliterated. There is a decrease in

intertubular and Bowman's capsular space with an increase

Of red blood cells in glomerular and peritubular capillaries.

 

Fig. 38. Rat, Control B.P. - 95 mm. Hg, Collodion and Gauze
Wrap and Contralateral Nephrectomy, Hypertensive B.P. - 178
mm. Hg, In Vivo Freeze—Dry Technique at 7 days, wright's, x300.
There is marked necrosis and foreign body reaction.
Eosinophilic staining is prevalent in the outer cortex.

Alternating areas of blood stasis and ischemia appear through—

out the section. Intertubular and Bowman's capsular space is

greatly decreased.

59

 

Fig. 39. Rat, Control B.P. — 110 mm. Hg, Collodion and Gauze
Wrap of 21 Day Hypertrophied Kidney, Hypertensive B.P. — 158
mm. Hg, In Vivo Freeze—Dry Technique at 7 Days, wright's, x300.

This shows little change from controls (see figure 36),

although some tubular lumina are obliterated.

60

 

Fig. 40. Rat, Control B.P. — 112 mm. Hg, Collodion and Gauze
Wrap Followed in 7 Days by Contralateral Nephrectomy, Hyper—
tensive B.P. — 165 mm. Hg, In Vivo Freeze—Dry Technique at 7

Days, Wright's, x300.

Tubular lumina, which are not stenosed, appear to be
distended greater than normal. As in figure 38, there are
alternating areas of blood stasis and ischemia. Intertubular

and Bowman's capsular space is minimal.

 

Fig. 41. Rat, Control B.P. — 125 mm. Hg, Bilateral Adrenal-
ectomy, In Vivo Freeze—Dry Technique at 4 Days, wright's,
x300.

Many tubules are shrunken with obliterated lumina.
There appears to be a small decrease in intertubular and

Bowman's capsular space. This appears similar to figures 28

and 34.

62

 

Fig. 42. Rat, Kidney Frozen After Excision, Cut Section
Allowed to Melt on warm Slide with No Lyophilization, wright's,
x300.

Appears similar to conventional histologic preparation,

although there are no open tubule lumina and Bowman's capsu-

lar spaces. Contrast to figure 35.

TABLE 2

NUMBER OF TUBULAR CROSS-SECTIONS AND PERCENTILE

AREAS OF RENAL CORTICAL SPACES

 

 

NUMBER OF %
TUBULAR % INTER-
CROSS- % TUBULAR % TUBULAR
ANIMAL SECTIONS GLOMERULI CELLS LUMINA SPACE
Fish 35 10 47 6 37
Frog 40 10 42 13 35
Turtle 78 8 49 10 33
Pigeon 178 ll 37 8 44
Mouse 102 ll 43 16 30
Rat 84 18 36 11 35
Guinea Pig 45 14 39 16 31
Rabbit 97 11 38 19 32
Cat 103 8 48 11 33
Dog 72 17 36 18 29
TABLE 3
SYMBOLS

ANIMAL - Animal used in experiment.

TREATMENT - Animal treatment before kidney freezing.

B.C.D. - Bowman's capsule mean diameter in microns.

G.D. - Glomerular tuft mean diameter in microns.

P.T.O.D. - Proximal tubule outside mean diameterin.microns.
P.T.I.D. - Proximal tubule inside mean diameter in microns.
D.T.O.D. - Distal tubule outside mean diameter in microns.
D.T.I.D. - Distal tubule inside mean diameter in microns.
H.L.O.D. - Henle loop outside mean diameter in microns.
H.L.I.D. - Henle loop inside mean diameter in microns.

63

TABLE 4

NEPHRON DIAMETERS IN.MICRONS FROM CONVENTIONAL
KIDNEY SECTIONS

 

 

 

 

No. ANIMAL B.C.D. G.D.
1. Fish 45 i 2.3* 37 i .
2. Frog 142 i 5.7 88 i
3. Turtle 115 i 4.0 109 i .
4. Pigeon 40 i 1.9 40 i
5. Mouse 67 i 2.0 61 i
6. Rat 112 i 3.2 99 i 4.1
7. Guinea Pig 136 i 3.7 120 i 3.
8. Rabbit 109 i 4.3 96 i .
9. Cat 95 i 2.1 83 i 2.0

10. Dog 120 i 3.7 98 i 3.6

NO. P.T.O.D. . P.T.I.D. D.T.O.D.
1. 45 i 12 i 1.5 - -
2. 74 i 4.0 48 i 3.6 64’i 2.3
3. 64 i .7 16 i 1.2 40 i 2.0
4. 36 i 1 i 0.8 24 i .
5. 40 i 4.7 0 i 0 40 i 3.4
6. 40 i 2. 3 i 0.8 38 i 3.1
7. 35 i . 8 i 1.2 43 i 3.9
8. 35 i 2.4 5 i 2.1 40 i 2.8
9. 40 i 2.2 13 i 4.2 29 i 3.3

10. 39 i 3.0 17 i 1.6 32 i 5.1

* Standard deviation

64

TABLE 4 (cont.)

NEPHRON DIAMETERS IN MICRONS FROM CONVENTIONAL
KIDNEY SECTIONS

 

 

ANIMAL
NO. D.T.I.D. H.L.O.D. H.L.I.D.
1. — - _ _ _ _
2. 40 i 3.6* 24 i 2.2 14 i 1.6
3. 17 i 1.1 21 i 3.9 6 i 1.1
4. 8 i 2.1 8 i 1.3 6 i .
5. 16 i 3.4 24 i 2.7 19 i .
6. 16 i 2.9 24 i 2.0 16 i .
7. 29 i 3.2 27 i 3.0 16 i .
8. 16 i 3.1 32 i 2.0 16 i 2.8
9. 16 i 2.8 22 i 2.5 13 i .
10. 16 i 1.8 22 i 1.9 12 i 2.4

 

* Standard deviation

65

TABLE 5

NEPHRON DIAMETERS IN MICRONS OF E VIVO FROZEN-DRIED KIDNEYS

 

 

TREATMENT

 

NO. ANIMAL B. C.D.
1. Fish None 56 i 1.4*
2. Frog NOne 193 i 5.9
3. Turtle NOne 135 i 5.7
4. Pigeon NOne 47 i 2.3
5. Mouse None 82 i 5.4
6. Rat None 126 i 4.1
7. Guinea Pig None 149 i 4.9
8. Rabbit None 126 i 6.3
9. Cat None 140 i 7.5

10. Dog None 151 i 8.1

11. Rat 15 sec. renal artery ligation 107 i 3.2

12. Rat 30 sec. renal vein ligation 126 i 3.0

13. Rat 4 minute ureter ligation 118 i 3.2

14. Dog 30 minute lymph duct ligation 126 i 5.9

15. Cat Renal capsule ablation 133 i 7.7

16. Rat 0.1 cc., 1:50,000 Adrenalin, I.V. 126 i 5.9

17. Rat 0.1 cc., 1:2,000 histamine, I.V. 110 i 4.5

18. Rat 0.5 cc., 20% glucose, I.V. 134 i 4.1

19. Rat 3 week hypertrophy 145 i 9.2

20. Rat Hypertension (method "A") 118 i 5.4

21. Rat Hypertension (method "8") 110 i 4.9

22. Rat Hypertension (method "C") 141 i 8.9

23 Rat Hypertension (method "D") 110 i 5.7

24. Rat 4 day adrenalectomy 102 i 5.1

25. Rat Remove kidney and drain 118 i 4.3

26. Rat Standard histochemical preparation 110 i 3.9

* Standard deviation

66

TABLE 5 (cont.)

NEPHRON DIAMETERS IN MICRONS OF IN VIVO FROZEN-DRIED KIDNEYS

 

 

 

67

* Mean inside diameter of tubules remaining

ANIMAL NO. G.D. P.T.O.D. P.T.I.D. D.T.O.D.
1. 56 i 2.6 47 i 3.7 14 i 2.2 — -
2. 180 t 71 i 39 t 3.3 71 f 4.3
3. 125 i 5.4 60 t 4.5 15 i . 55 i .
4. 39 i 28 i . 13 i 1.4 24 t 3.2
5. 68 i 4.5 25 i 13 t 30 i 3.2
6. 110 i . 31 t .6 14 i 3.4 35 i 3.9
7. 118 i 6.2 44 t 27 t . 49 t 4.1
8. 110 i 5.2 41 i 6.4 24 i 47 t 4.8
9. 118 i . 47 i 5.8 23 t 2.4 39 t 5.0

10. 126 i 4.3 39 t . 20 i 2.2 39 t 4.5
11. 107 i 31 t 4.1 0 — 33 i 3.6
12 118 i 3.6 35 t 0 - 47 t 4.9
13. 110 t 3.8 39 t 15 t 2.3 39 i 4.2
14. 126 t 5.9 '35 i. 0 — 33 i 2.7
15. 97 t 8.0 44 t 5.4 31 t 3.4 47 t 4.4
16. 110 t 39 t . 24*t 7.8 31 i 2.9
17 110 i 4.5 31 t 4.5 0 - 31 t 3.6
18. 113 i 7.1 42 i 5.7 28 _ 4.4 38 i 2.9
19. 126 t 9.3 47 t 31*i 6. 42 t 5.3
20. 110 t 5.6 39 t 4.7 27*t .2 50 t 3.4
21. 102 t . 31 t 3.6 22*: 6.4 44 t 6.6
22. 125 t 8.4 39 t 24*t 31 t 5.1
23. 94 t 42 t 4.3 25 t 3. 39 t 4.4
24. 97 t . 36 t . 18*t 2.1 19 t 1.8
25. 118 t . 39 t 3.5 0 - 30 t 2.8
26. 110 t 47 t 3.6 0 - 31 t 2.6
open

TABLE 5 (cont.)

NEPHRON DIAMETERS IN MICRONS OF IN VIVO FROZEN—DRIED KIDNEYS

 

 

 

ANIMAL NO. D.T.I.D. H.L.O.D. H.L.I.D.
1. — - _ _ _ _
2. 39 — 3.1* 42 - 4.2 31 i 3.8
3. 8 i 1.7 19 i 2.3 8 i 2.1
4. 17 i 2.6 13 i 2.1 9 i 2.8
5. 14 i 2.5 22 i 13 i 2.0
6. 17 i 2.8 23 i 2.4 19 i 1.9
7. 28 i 3.3 24 i 2. 15 i 2.2
8. 34 i 4.2 20 i 3.5 14 i 2.9
9. 20 i 3.1 23 i 3.4 16 i 1.7

10. 20 i 3.4 27 i 2.1 17 i 2.3
11. 16 i 3.8 19 i 2.9 13 i 2.5
12. 27 i 4.0 31 i 3.2 27 i 2.8
13. 22 i 3.5 31 i 3.3 14 i 2.3
14. 16 i 2.4 14 i 6 i 2.2
15. 31 i 3.0 24 i 3.4 19 i 3.2
16. 16 i 2.3 24 i 2.5 9 i 1.7
17. 16 i 3.3 19 i 1.6 11 i 1.3
18. 31 i 2.4 24 i .1 16 i .
19. 20 i 3.6 36 t 4.1 24 i 3.4
20. 28 t 3.3 31 i 20 i

21. 30 i 5.3 30 i 3.8 20 i 2.9
22. 22 i 4.6 35 i 3.7 25 i 3.3
23. 24 i 4.2 24 i 2.3 19 i 2-5
24. 10 i 1.6 27 i .8 19 i .
25. 0 — 23 i 2.4 16 i 2.4
26. 0 - 19 i 1.7 6 i 1.

 

* Standard deviation

68

69

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DISCUS SION AND CONCLUSIONS

Although it has limitations and could be improved, the

in vivo freeze—drying technique offers scientific investi-

 

gators a tool for a better insight to cellular anatomy and
function. Some of the limitations to be considered are: the
cryostat—microtome gives a variable tissue section thickness

at any one setting; In_vivo frozen-dried kidney sections show

 

red blood cell shrinkage and intercellular membrane separa-
tion, thus there is some evidence for distortion; cellular
and nuclear membrane tearing may result from handling of
dried tissues; and wright's stain shows different dye absorp—
tion characteristics by cells in sections from various
species despite the same procedure and timing. Among the
advantages of the technique are: prevention of post mortem
diffusion and osmosis often encountered with other techniques;
elimination of cellular autolysis; and nearly instant arrest
of cells in the living state thus preserving the functional
anatomical relations.

In the comparative study, as shown in figures 7 through
26, larger tubular luminal and intertubular spaces are pres-
ent in the In yInn_frozen-dried kidney sections (figures 8,

10, 12, 14, 16, 18, 20, 22, 24, and 26) than in sections

74

75

from conventional histologic methods (figures 7, 9, 11, 13,
15, 17, 19, 21, 23, and 25). The differences are smaller in
fish, frog, and turtle, which have lower mean arterial pres-
sures than in other species studied. Kidney sections by
conventional methods are red cell poor because of post mor-
tem drainage prior to fixation. Swann 2E.él- (1955, 1956 a & b,
1958 a & b, 1960, 1962) have extensively reported on the
fluid loss from the freshly excised kidney. Nephron diam-
eters of In nyn_frozen-dried kidney sections are 1 to 51
microns larger than sections from the conventional histo-
logic methods. With conventional histologic methods tubular
luminal diameters and loss of interstitial space could re—
sult from the fixation procedure and osmotic swelling.

The interdigitating nature of tubular cells with In_yInn_
freeze-drying, as shown in figure 27, could be a result of
freezing or lyophilization. Definite cellular spacing leads
one to believe that considerable shrinkage or selective ex-
pansion occurred. Then too, this might be natural tubular
cell conformation between apical and basement membranes. If
the cell membranes do intususscept or interdigitate, as shown
in figure 27 and by Pease (1955b & c), this would be an
advantageous arrangement leading to increased intercellular

surface area.

76

Using Brodie's (1941 ) form of Poiseuille's equation for
fluid fkwv(pressure = 1.611 x 104 ° L/r4 mm. Hg) and 12.2i12
froze -dried kidney section tubular measurements, the mini-
mum pressure required to force filtered urine through the
nephron length is 9.33 mm. Hg in dogs and 11.91 mm. Hg in
rats for a flow of 1 ml. per minute. This coincides fairly
well with the 13.5 mm. Hg mean tubular pressure of rats as
directly measured by Gottschalk and Mylle (1956).

Rat renal artery ligation 15 sec. before freezing (fig-
ure 28) produces a virtual obliteration of proximal tubular
lumina and intracapsular space of Bowman's capsule. In addi-
tion there is a significant* decrease in the following
diameters: Bowman's capsule (figure 43), glomerulus (figure
44), inside diameter of the proximal tubules (figure 45),
and inside and outside diameters of Henle's loop (Table 5).
Even though the proximal tubular inside diameter drops to
zero (Table 5, figure 45), the outside diameter shows no
significant difference from controls, and intertubular space
remains abundant. Apparently, sufficient glomerular hydro-

static pressure and/or glomerular blood flow are necessary

* Unless stated otherwise, significance in this discussion
is at the 1%.leve1 as determined by the t distribution.

77

for maintaining capsule distance from the glomerular tuft.
Blood pressure and flow are probably also necessary to main-
tain proximal tubular lumina. Proximal tubular luminal
obliteration, as seen here, may be the overextension of the
brush border as suggested by Pease (1955a & b).

Rat renal vein ligation 30 sec. before fixation by freez-
ing (figure 29) produced marked red cell packing in peri-
tubular and glomerular capillaries. No significant differ-
ence is shown with Bowman's capsule diameter (figure 43),
although intracapsular space is diminished by significant
glomerular expansion (figure 44). As with renal arterial
ligation, tubular lumina are stenosed and intertubular space
is abundant. Here, glomerular blood pressure is probably
higher because of venous occlusion. Hewever, glomerular
blood flow is likely diminished and, as a result, filtration
is minimal and tubular closure ensued, probably due to stasis
of filtrate and its osmotic passage into the tubule cells.

Four minute rat ureter ligation (figure 30) increased
significantly outside tubular and glomerular diameters and
produced red cell packing in glomeruli and peritubular capil-
laries. This could have occurred as follows: ureter liga-
tion raises ureteral resistance to the maximum, and the

raised ureteral pressure was transmitted to the tubules via

78

the collecting ducts. The tubules would dilate into the
intertubular spaces (Gottschalk, 1952, found an increase in
intertubular pressure with ureteral ligation), collapse the
blood vessels causing high resistance to blood flow, in-
crease glomerular blood pressure, and hence, greater glomer-
ular diameter (figure 44). Again, as with vein ligation,
renal blood flow is diminished (Winton, 1951; Hinshaw gn_nI,,
1959; and Hinshaw g; nI., 1961), thereby decreasing glomeru—
lar filtration and, as a result, proximal tubule stenosis
ensues (figure 45).

As shown in figure 30, thirty minute renal lymph duct
ligation in the dog produces 45% increase in intertubular
(interstitial) spaces. Lymph ducts are blind endothelial
tubes, which terminate in the interstitium and partly func-
tion to relieve excess interstitial pressure consequent to
filtration from capillaries. Here, lymph vessels draining
the interstitial areas are obstructed and capillary filtra—
tion results in accumulation of fluid in the extracellular
space and increased interstitial pressure. As hypothesized
by Haddy__§_nI. (1958), this pressure increase could cause
peritubular capillary collapse and decreased renal blood

flow. The resultant decreased glomerular filtration could

cause, as observed in figures 30 and 46, proximal tubular

79

stenosis. In addition, as shown in Table 5, there is a
significant decrease in outside diameters of all tubules.

As seen in figure 32, cat renal capsule removal produces
tubular distension and stretching (compare to figure 24),
Bowman's capsule and glomerular diameters are significantly
decreased (Table 5), although intracapsular space (Bowman's
capsule) is larger. Proximal (figure 47) and distal tubular
inside diameters are significantly increased. These results
are consistent with Brodie's (1914 ) findings in which he
reported that renal capsule removal in the living cat pro-
duced an increase in tubular luminal and intracapsular
(Bowman's capsule) spaces. One function of the renal capsule
is probably to prevent overdistension of the turgid organ.

Intravenous adrenalin chloride (0.1 m1., l:50,000) in
rats produces some signs of kidney tubule stenosis (figures
33 and 45) and space depletion in about half of Bowman's
capsules in a section. There is no significant change
(Table 5) in glomerular (figure 44) or Bowman's capsular
(figure 43) diameter, although intertubular space is de-
creased. The initial adrenalin slug probably acted prima—
rily on the afferent glomerular arteriole (Ahlquist g; QI.,
1954) to drop glomerular blood flow and filtration pressure

causing proximal tubular stenosis. Later, the diluted,

8O

recirculating adrenalin dilated the afferent and constricted
the efferent glomerular arterioles (Richards and Plant, 1922
a & b). Subsequently, glomerular filtration pressure in-
creased over the original and increased tubular pressure
caused significant tubular distension. Since, at least one
minute elapsed from injection to fixation by freezing, the
slides probably represent the second phase of the adrenalin
action in which the nephrons are recovering from the initial
glomerular blood flow drop.

Intravenous histamine diphosphate (0.1 m1., 1:2,000) in
rats produces proximal tubular stenosis (figures 34 and 45)
and a marked drop in Bowman's capsular space. The result is
very similar to renal artery ligation (figure 28), although
with histamine interstitial space is less and red blood cells
seem to be more abundant in capillaries. Also, there is a
significant difference from controls in glomerular (figure
44) and Bowman's capsular (figure 43) diameters with hista-
mine producing enlargment of both structures. Histamine
causes a drop in systemic pressure (Bjering, 1937), and
renal vasodilation (Page and McCubbin, 1953). Again, de-
creased glomerular blood flow and/or decreased glomerular
blood pressure apparently caused proximal tubular narrowing.
Renal blood flow, although apparently minimal, is adequate

as indicated by normal glomerular size (figure 44) and the

81

red blood cell filled capillaries.

Diuretic rat kidneys (figure 35) show a significant in-
crease in Bowman's capsular (figure 43), glomerular (figure
44), and internal (figure 45) and external tubular diameters
(Table 5). The organ appears to be porous. Hypertonic
glucose, used to produce diuresis, undoubtedly increased
filtration and tubular pressures and caused tubular disten-
sion. The epithelium is thinned by stretching and there is
little increase in brush border extension. Gottschalk and
Mylle (1956) indicate this overall process when they reported
increased intratubular pressure after hypertonic glucose
injection.

After three weeks of hypertrophy, rat kidneys (figure 36)
appear to be similar to controls but there are significant
increases over controls in all nephron diameters (figures
43, 44, and 45, Table 5). This increase is probably due to
cellular hypertrophy and/or cellular hyperplasia; no visual
distinction can be made between the two processes.

Hypertensive rat kidneys (figure 37), produced by renal
artery constriction (method "A“), show some stenosed tubules,
a decrease in Bowman's intracapsular and intertubular space,“
and erythrocyte filled capillaries. Nephron diameters are

significantly decreased over controls (Table 5, figures 43,

82

44, and 45) except distal tubules, and these are signifi-
cantly greater than controls. Pearse (1961) believes the
distal tubule is intimately concerned with renal hyperten-
sion as it shows a glucose-6-phosphodehydrogenase increase
in distal tubules during early stages of the disease. The
tubular hypertrophy is thought to indicate increased enzyme
production.

Simultaneous collodion and gauze kidney wrap and contra-
lateral nephrectomy (method "B") in rats (figure 38) produces
striking, rapid results. Marked necrosis and foreign body
reaction, from the collodion and gauze, prevails in the
outer cortex. There are areas of both blood stasis and
ischemia and the intertubular area is scanty. Nephron diam-
eters (Table 5) are significantly less than hypertrophied
controls, except the distal tubule diameter, and it is sig-
nificantly greater than controls. Apparently, tubular hyper—
trophy into intertubular spaces, as theorized by Swann $2.9;-
(1959), pinches peritubular capillaries, raises renal blood
flow resistance, and promotes renal ischemia.

Collodion and gauze wrap of hypertrophied rat kidneys
(method "C") shows little difference from hypertrophied
controls (compare figures 36 and 39), although there is a

significant decrease in outer and inner proximal tubular and

83

outer distal tubular distance.

As shown in figure 40, hypertension in rats by collo-
dion and gauze wrap followed seven days later by contra—
lateral nephrectomy (method “D") produces a significant de-
crease (Table 5) in Bowman's capsular and glomerular diam-
eters (figures 43 and 44). Intertubular space is decreased
over hypertrophied kidneys and, as in method "B,” there are
areas of both blood stasis and ischemia. Tubular diameters
are significantly less than hypertrophied controls. How-
ever, distal tubule inner diameter is significantly greater
than hypertrophied controls.

Comparing the results of hypertension production, there
appears to be a direct correlation between the restriction
of renal distension and blood pressure increase over the
control. An estimated twenty percent renal artery constric-
tion by a silver clamp (method "A") caused a 28 mm. Hg sys-
tolic blood pressure increase. Collodion and gauze wrap of
a hypertrophied kidney (method "C") causes a moderate dis-
tension restriction and a systolic blood pressure increase
of 48 mm. Hg. Simultaneous collodion and gauze kidney wrap
and contralateral nephrectomy (method "B") produces a severe
distension restriction and a systolic blood pressure increase

of 83 mm. Hg. However, if, as in method ”D," one waits 7

84

days before removing the contralateral kidney, allowing the
organ to remain and carry a portion of the excretion burden
while the body and the wrapped kidney adjust to the collo-
dion and gauze, the systolic blood pressure increase is only
53 mm. Hg. This indicates that perinephritis and restricted
renal distension cause more pronounced renal hypertension
than restricted renal distension alone.

Kidney sections from four-day adrenoprival rats (figure
41) show decreases in intertubular and proximal tubular
luminal space. Bowman's capsular space is minimal. Bow-
man's capsular and glomerular diameters are significantly
less than controls (figures 43 and 44). Proximal tubular
and Henle's loop diameters are significantly greater than
controls, although distal tubule diameters are significantly
less than controls. Apparently, the conditions of hypoten-
sion and hypovolemia, as stated by Turner (1955) in adrena-
lectomized animals, produce these findings.

As seen in figure 42, rat kidneys, which were drained,
frozen, cut, melted, dried, and stained, appear similar to
conventional histologic slides (figure 17). Although very
small intertubular and tubular luminal spaces were present,
nuclear and cytological detail are unclear. In comparison

to In vivo frozen—dried controls (figure 18), it appears

 

85

that considerable osmosis and diffusion could have taken

place.

In conclusion, since the technique used here indicates

the necessity for some revision in the classical, textbook

concept of functional renal histology, it is suggested that

these procedures might be applied profitably to the study

of other organs.

SUMMARY

1. Kidneys of ten animal species were frozen, In_nyg,
with -1750 C. isopentane, cut, lyophilized before thawing,
stained, with alcoholic wright's, and mounted; and contra—
lateral kidneys were processed by conventional histologic
methods. Comparison revealed marked differences with
frozen-dried kidneys exhibiting wide tubular lumina, thin
tubular epithelium, and large intertubular areas; and inter—
digitating cells appeared in larger tubule walls.

2. Ligation of rat renal artery, vein, or ureter or dog
renal lymph ducts disturbs delicate intrarenal pressure
relations to cause blood stasis and proximal tubular luminal
stenosis. Renal lymph duct ligation also causes marked
intertubular area increase.

3. Cat renal capsule removal produces tubular stretching
indicative of overdistension. This is consistent with ear-
lier findings. Apparently, the renal capsule prevents over-
distension in the normal functioning state.

4. Concentrated doses of adrenalin or histamine in rats
produce renal tubular luminal stenosis, but the secondary
adrenalin effect, upon recirculation, produces tubular dis-

tension. Glucose, acting as a diuretic, significantly

86

87

distends and stretches tubules more than in "resting"
kidneys.

5. Twenty-one day hypertrophied rat kidneys have signi-
ficantly greater nephron diameters than controls. No
distinction can be made between growth by simple hyper-
trophy or hyperplasia.

6. Seven day hypertension by renal artery constriction or
perinephritis causes tubular luminal stenosis, a decrease
in Bowman's intracapsular space and intertubular space, and
blood stasis to a varying extent depending on the method
used.

7. Four day adrenoprival rat kidneys showed decreased in-
traluminal and intertubular space. Apparently, hypotension
and hypovolemia, associated with the syndrome, cause this
condition.

8. Drained, frozen, cut, melted, and stained rat kidneys
as prepared in this study, appear similar to the usual histo-
logic preparations. However, the true compartmental rela-

tionships are distorted.

REFERENCES

Ahlquist, R. P., Taylor, J. P., Rawson, C. W., and Sydow,
V. L.: Comparative Effects of Epinephrine and Levor-
terenol in the Intact Anesthetized Dog. J. Pharmacol.
& Exper. Therap., 110:353-360, 1954

Altmann, R.: Die Elementarorganismen und ihre Besiehungen
zur den Zellen. Veit & Co., Leipzig, 1890

Andrew, W.: Textbook of Comparative Physiology. Oxford
University Press, New York, 1959

Arcadi, J. A., and Tessar, C.: A Simplified Altmann-Gersh
Freezing-Drying Apparatus. J. Lab. Clin. Med., 43:
479-481, 1954

Babin, D. 8., Robbins, G. B., Turgeon, C., and Sommers,
S. C.: Kidney Maps in the Study of Human Renal Disease.

Fed. Proc., 20:408, 1961

Bayliss, W. M.: Principles of General Physiology. Longmans,
Green and Co., London, 1915

Bell, L. G. E.: The Application of Freezing and Drying
Techniques in Cytology. Int. Rev. Cytol., 1:35-63, 1952

Bell, L. G. E.: Physical Techniques in Biological Research.
(Oster, G. and Pollister, A. W., eds.) Academic Press,
New York and London, 1955

Bjering, T.: The Influence of Histamine on Renal Function.
Acta. med. Scandinav., 91:267—278, 1937

Bloom, W. and Fawcett, D. W.: A Textbook of Histology. W. B.
Saunders & Co., Philadelphia and London, 1962

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