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MEDICAL LIBRARY

Digitized by the Internet Archive
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https://archive.org/details/effectofamphoterOOklee

THE EFFECT OF AMPHOTERICIN B ON THE

PROXIMAL TUBULE OF THE RAT KIDNEY

by

Stuart R. Kleeman
A. B. Brown University 1967

A thesis submitted to the
Department of Internal Medicine
Yale University School of Medicine
in partial fulfillment
of the requirements for the degree of
Doctor of Medicine

April 1971

New Haven, Connecticut

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ACKNOWLEDGMENT

To Dr. John P. Hayslett, for his guidance, patience and advice
in many matters for the past four years .

To Dr. Michael Kashgarian, for his instruction, both technical
and theoretical, and the demonstration that mastery of the machinery
is possible.

To Dr. Edward J. Weinman, for his constant encouragement.

To Dr. Franklin H. Epstein, for his inspiration and the intro¬
duction to my other mentors.

To Mr. Richard Whittaker and Mrs. Dorothy Smith, for their
valuable technical instruction and assistance.

To Mrs. Lena DelCorte, for her secretarial assistance.

And finally, to my wife, Terry, for her devotion, confidence
and an uncanny ability to help me past my minor disasters.

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TABLE OF CONTENTS

Introduction . 1

Materials . 2

Methods . 5

Results . 15

Discussion . 21

Summary . 41

References . 42

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INTRODUCTION

In the field of renal physiology, many investigators have recently
been attempting to evaluate and clarify the mechanisms which control the
reabsorption and the ultimate urinary excretion of sodium. These mech¬
anisms are vital to fluid and electrolyte homeostasis, for by varying
the excretion of sodium, the principle cation of the extracellular fluid,
the organism is able to influence the volume of this fluid compartment
and control for differing levels of sodium intake.

Among the parameters under investigation in the control of sodium
reabsorption are circulating hormones, changes in peritubular physical
factors and the distribution of renal blood flow to functionally dif¬
ferent parts of the renal cortex. In order to be accessible to the
influence of these factors, however, sodium must cross the luminal mem¬
brane into the epithelial cell and be available to the sodium pumping
apparatus. The effect of possible variations in luminal membrane perme¬
ability has been, thusfar, relatively neglected.

-This study was an attempt to clarify the role of this barrier in
sodium reabsorption by altering its permeability. Using micropuncture
techniques , the lumen of the proximal tubule of the rat kidney was per¬
fused with amphotericin B, a polyene antibiotic known to increase the
permeability of membranes in many in vitro systems , and the changes in
sodium and fluid reabsorntion were measured.

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MATERIALS

1 . Experimental Animals . Adult male Sprague-Dawley rats weighing
220-410 gm were obtained from Camm Research, Wayne, N.J., and Charles
River, No. Wilmington, Mass. They were maintained in individual cages
on Purina Chow and tapwater, ad libitum. Food and water were not re¬
stricted prior to the induction of anesthesia at the time of each
experimental study.

2. Construction of micropipettes. Double-barreled micropipettes
were prepared from glass capillary tubing, approximately 1 mm in external
diameter that had been washed in concentrated sulfuric acid and air
dried. Two 15-20 cm lengths of tubing were manually pulled and twisted
over a small flame resulting in union of the two in a pattern of 2-4
tight spirals. The tubing was heated at the point of union and pulled
apart by an automatic micropipette puller (Industrial Science Associates,
Inc., Ridgewood, N.Y.). In this way, a double-barreled micropipette was
obtained with a common, tapered tip. At the mid-point of the micro¬
pipette, one barrel was gently heated and manually bent to an angle of
80°, so that each barrel could be individually filled. The tip of each
micropipette was viewed at 100 X under a monocular microscope fitted
with a filar eyepiece micrometer and manually broken off with a forceps
to an outside diameter of 7-10 y. The micropipettes were held in a
pipette holder on a micromanipulator (Model 5003, Brinkmann Instruments,
Inc., Great Neck, N.Y.), and the tips were beveled against a carbor¬
undum grinding wheel (Model 37C 320 LIIV, Norton Abrasives, Teterboro,
N.Y.) to an angle of approximately 30 . Each micropipette was examined
microscopically to guarantee a smooth, patent, beveled tip of appropriate

dimensions .

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3. Solutions and Routes of Administration

A. Anesthesia. Inactin (Chem. Fabrik. Promonta, Hamburg, Germany)
was made up daily in 154 mMNaCl at a concentration of 80 mg/ml. Rats were
injected intraperitoneally at a dosage of 160 mg/kg, body weight. This
agent produced a stable state of general anesthesia which was maintained
for the duration of the acute experiments (2 to 4 hours). Additional
increments of anesthetic were not required.

B. Infusion Solutions

Intravenous solutions were administered via indwelling poly¬
ethylene catheters placed in the external jugular veins.

i. 154 mMNaCl was made up in distilled water and used to replace
surgical losses and provide maintenance hydration during the entire
course of each experiment.

ii. 10% Lissamine green was made up in distilled water and in¬
jected periodically to measure proximal tubule transit times.

Intratubular- The double-barreled micropipettes were filled just
prior to use with an aqueous test solution in the bent arm and a filtered
suspension of Sudan black (for photographic contrast) in castor oil in
the straight arm. The test solutions consisted of:

i. 154 mMNaCl

ii. Amphotericin B, as the solubilized Fungizone preparation
(E.R. Squibb and Sons, New York, N.Y.), was dissolved in 154
mMNaCl to final concentrations of:

amphotericin B, 50 yg/ml
sodium desoxycholate , 41 yg/ml
sodium phosphate buffers, 25 yg/ml.

These amounts resulted in an addition of 0.97 mOsm/Liter to the
isotonic saline (a 0.32% increase in osmolarity).

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iii. A third solution containing all the constituents of Fungi¬
zone with the exception of amphotericin B was made up in 154 mMNaCl
to the same concentration as in the amphotericin B test solution
(ii).

Solutions ii. and iii. were prepared weekly and refrigerated at 4°C
until used.

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5

METHODS

1. Surgical Preparation

After the induction of anesthesia with Inactin, the neck, abdo¬
men, and left flank were shaved. The animal was placed supine on an
electrically heated board with the temperature controlled by a type 116
Powerstat (Fisher Scientific, New York, N.Y. ) and body temperature mon¬
itored with an esophageal temperature probe (Tele-thermometer, VS1,
Yellow Springs Co., Inc., Yellow Springs, Ohio) and maintained at 37°-
38°C .

A tracheostomy was performed with a 3-4 cm segment of tapered Clay
Adams PE 260 polyethylene tubing inserted in the trachea, secured with
3-0 surgical silk and cut as short as practicable to limit respiratory
dead space. One external jugular vein was cannulated with Clay Adams
PE20 polyethylene tubing filled with 154 mMNaCl and secured with 4-0
surgical silk. The urinary bladder was exposed through a suprapubic
incision, and a short length of Clay Adams PE50 polyethylene tubing
with a heat-flanged tip was secured in the bladder with 3-0 surgical
silk .

A vertical midline incision was made in the abdominal skin and the
recti divided from approximately 1 cm cephalad to the bladder incision
to the xiphoid process. A left subcostal incision was carried from the
midline to the posterior axillary line, and the animal was moved onto
its right side. Overlying loops of bowel were gently retracted from
the left upper quadrant, and the left kidney was visualized.

Using two pairs of small forceps, the perinephric fat and fibrous
renal capsule were carefully stripped away from the superficial paren¬
chyma leaving the entire cortical surface exposed (1). The kidney was
placed in a small rectangular plexiglas cup which contained a broad

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notch on one side to accomodate the renal pedicle in order to avoid
vascular embarassment to the kidney. The cup was lined with cotton
fluff soaked with mineral oil to immobilize the kidney and reduce
respiratory movement and vascular pulsations. A brass handle attached
to the cup was secured in a thumb-screw holder on the animal board,
and the angle of the handle and the cotton packing were adjusted so as
to leave the surface of the kidney as close to horizontal as possible.
The skin and muscle edges were retracted with modified safety pins
anchored to the cork surface of the animal board, and the kidney was
covered with mineral oil, at room temperature, which was replenished at
ten minute intervals.

154 mMNaCl, in an amount equal to 1% of the animal's body weight,
was infused over an 8-12 minute period by a Compact Infusion Pump
(Model 975, Harvard Apparatus Co., Inc., Dover, Mass.) to replace sur¬
gical fluid losses. A 1.2 ml per hour infusion was maintained for the
remainder of the experiment .

The animal, lying on the heated animal board, was placed on an
adjustable stage (Big Jack, Precision Scientific Co., Chicago, Ill.)
under a stereomicroscope (Model 570, 0.7-4. 2 X, American Optical Co.,
Buffalo, N.Y.). The entire exposed surface of the kidney was visual¬
ized at 84 X with illumination from a 100 watt zirconium arc lamp with
heat reflecting glass filters (Paul Rosenthal, Inc., Great Neck, N.Y.),
and individual surface tubules could be clearly visualized.

2. Experimental Design

Experiments lasted from 2-4 hours, including the time required
for surgical preparation. During the course of each experiment, prox¬
imal tubule transit times were estimated at 20-30 minute intervals to
assess the viability of the preparation. From one to six proximal

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tubules were selected on the basis of criteria for length, shape, and
accessibility by the micropipette, and each tubule was blocked with
castor oil, perfused with an aqueous solution and photographed no more
than twice. During the course of the reabsorption of each aqueous
droplet, from three to six photomicrographs were taken at three second
intervals, and at the conclusion of each experiment, the film was
developed, enlarged, and printed under controlled conditions. All
prints were grouped into ordered sequences of individual droplets, and
each sequence received a random code number. All droplets were measured
and reabsorptive half-times (t%) were calculated without the observer
knowing the composition of the droplet under consideration. After the
calculation of all t%s , the code was revealed and the droplets were
grouped according to the nature of the test solution. Statistical
analysis was performed, and the differences in the calculated mean t^s
were compared using Student's t test.

3. Proximal Tubule transit times.

In order to evaluate the condition of the preparation as a whole
and test the patency of individual proximal tubules, a modification of
Steinhausen' s (2) method for distinguishing proximal from distal tubules
on the kidney surface was employed .

When viewed through the microscope with proper illumination, prox¬
imal tubular loops can be distinguished from the much rarer distal
tubular loops by the bright band, representing the brush border, on
the luminal surface of cells of the proximal convolution, which is
virtually absent in the distal tubule. Only the middle third of the
proximal convoluted tubule is present on the kidney surface, and the
terminal superficial segments of this portion of several proximal
tubules are seen to converge in groups surrounding single efferent

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arterioles in a series of "star" or "rosette" conformations. This con¬
formation represents the point at which tubular fluid has traversed 55-
60% of the proximal tubule and is about to desend into the cortex in the
pars recta (3).

To estimate proximal tubule transit time, the surface was observed
while 0.5 ml of 10% Lissamine green was rapidly injected intravenously
with a gastight syringe (#1002, Hamilton Co., Whittier, California).
According to the modification of Gertz, et al. (4), the transit time was
measured from the occurrence of the initial "green blush" of the renal
surface, resulting from the filling of surface vasculature and Bowman’s
capsule and indicating the entrance of dye into the proximal tubule, to the
time when the filtered dye had filled the last of a group of tubules form¬
ing a "rosette". These intervals which were usually 9-11 seconds were
used as the index of viability of the kidney. If and when this transit
time became grossly prolonged (greater than 15 seconds), the experiment
was terminated.

4. Stationary Microperfusion.

Purpose. The technique of stationary microperfusion of proximal
tubule segments, also known as the "split oil droplet" method, was used
to evaluate net fluid efflux under control and experimental conditions.

Theoretical Formulation. In the original description of this method,
Gertz (5) demonstrated that when a column of oil filling the proximal
tubular lumen was split by a droplet of saline, the progressive net
reabsorption of the saline was accompanied by the convergence of the
ends of the oil columns. The regression in the length of the droplet,
therefore, was considered to be directly proportional to the rate of
fluid reabsorption. The radius of any given droplet was considered to
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droplet shrinkage. The relationship between the length of the droplet

and time was demonstrated to be semilogarithmic and can be expressed

as: (1) In / 1 (t ) \ = mt

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where 1 (t) = the droplet length at time, t,

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Procedure. The double barreled micropipettes were filled to the
tips with castor oil and an aqueous test solution and placed in a Leitz
micromanipulator. A compressed air line was connected to the straight
arm containing the castor oil, and a 50 ml glass syringe was attached
via, polyethylene tubing to the bent arm with the aqueous solution. The
kidney surface was scanned in order to locate a segment of proximal
tubule which appeared to be grossly straight, with paralled sides,
totally on the surface, approximately 3-5 tubular diameters in length,
and aligned approximately in the orientation of the micropipette. The
micropipette tip was moved as close to the end of a length of tubule as
possible, and lowered at an angle between 15° and 40° to the horizontal,
far enough to barely depress the tubular epithelium. The tubule was
punctured with a short, brisk motion along the axis of the micropipette
leaving the openings of both barrels entirely within the lumen of the
tubule. Castor oil was injected in amounts sufficient to block tubular
flow, and the oil was followed by the infusion of the test solution drop¬
let. It was usually necessary to move the aqueous droplet away from the
pipette tip by injecting more oil.

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The sequential decrease in the length of the droplet, as reabsorp¬
tion progressed, was recorded by time-sequence photomicrography, per¬
formed on Tri-X film (El 1200, Eastman Kodak, Rochester, N.Y.) with a
Nikon F 35 mm camera with electric motor drive (Model F-36) mounted on
the ocular of the microscope. The camera and a home-made flash illu¬
minator, powered by a battery pack, were triggered at three second
intervals by a Nikon intervalometer (Model NCI) until manually terminated
when the droplet was reabsorbed. The exposed film was developed in
Acu-1 film developer (Acufine, Chicago, Ill.), diluted 1:5 in water,
for eight minutes at 75°F, enlarged and printed on Kodabromide F-4
paper (Eastman Kodak, Rochester, N.Y.). All prints were made at the
same magnification (Fig. 1).

5. Droplet Measurement.

In order to reduce observer bias , the prints were grouped as
sequences of individual droplets, and each sequence given a randomly
selected code number. The photographs were saved until all the drop¬
lets could be measured at a single sitting, without the observer having
the knowledge of the composition of the droplets.

The measurements were performed using a pair of drafting dividers.

The shortest distance between the menisci of the oil columns was measured,
compared with an arbitrary scale, and recorded as the droplet length, 1,
for each time, t, (0, 3, 6, 9, 12 seconds, etc.) as seen in Table 1.

The slope of the regression of length for each time interval was
calculated with the Programma 101 digital computor (Underwood Olivetti,

New York, N.Y.) using formula (2). The slopes for all time intervals
were averaged, yielding the mean slope of each droplet sequence. When
the original droplet has shrunken to one-half its initial length,
is equal to 0.5. The time required for this degree of reabsorption is

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defined as the reabsorptive half-time, t^. By rearranging formula (1),
and substituting the appropriate values,

(3 ) In (0.5) =

m

and since In (0.5) = -0.693

(4) th = -0.693 .

m

Using formula (4), a t% for each droplet was calculated. The code
was broken at the conclusion of the droplet measurements and calcu¬
lations, and the droplets were grouped according to their composition.

6. Statistical Calculations.

The t^s were plotted against the initial droplet lengths, 1 (o)
to determine whether there was a correlation between these factors which
might bias the results. A coefficient of correlation was calculated for
the saline control data and recalculated after arbitrary limits had been
placed on 1 (o) to provide similar distributions of 1 (o) for all experi¬
mental groups. The mean, standard deviation and standard error of each
revised group (with limits on 1 (o)) were calculated, and the mean th
for each group was compared with the other two groups using Student’s t
test to determine the significance of calculated differences.

In order to express the reabsorptive capacities in more quantita¬
tively descriptive terms than t^, the net flux of water was calculated
(6). Water flux (Jv) is equal to the change in volume per unit surface
area per unit time. If the droplet is considered a regular cylinder
with its radius expressed as r:

(5) Jv = (ur^) dl ,

(2 Trr lo) dt

where lo = initial length of droplet

t = time (seconds).

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14

Integration of expression (5) gives:

(6) Jv = In / 1_\ . r_

\ lo ) 2t

If ts th9 In = In (0.5) = -0.693

and substituting in (6):

(7) Jv = -0 . 347r .

t^

The actual tubular radius was established by measuring oil filled

tubules with a stage micrometer photographed at the same magnification

as the droplets. By substituting the calculated mean tubular radius

and t^ for each group into formula (7), the net water flux for each

3 2

experimental group was obtained and expressed as mm /mm" per second.

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15

RESULTS

1. Analysis of the stationary microperfusion method as an estimate
of net sodium reabsorption - The relationship of initial length and
reabsorptive half-time.

All of the acceptable droplets (straight, parallel lengths of prox¬
imal tubules, entirely on the surface) were measured and t^s were
calculated for all the experimental groups. Since the saline control
group had the fewest experimental manipulations, by virtue of the
simplicity of the composition of the droplet, it was elected to evaluate
the effect of initial length on reabsorptive half-time in this group.

When these t%s were plotted against 1 (o), some impression of the effect
of initial droplet length on the calculated reabsorptive capacity
becomes readily apparent (Fig. 2). Merely by inspection, it can be
appreciated that at shorter 1 (o), t^g tends to be of much shorter
duration. A coefficient of correlation for these 40 original droplets
from 14 different experimental animals over a range of initial lengths
of 6 to 19.5 units was calculated to be r = 0.4708 which was significantly
positive at p<0.01. Since in each of the three groups, there was a sim¬
ilar distribution of initial lengths between 9 and 20 units, these limits
were arbitrarily placed on the range of initial droplet length. The
coefficient of correlation was recalculated for this revised group of
33 droplets at r - 0.2755 which was not significantly positive with
p>0.1 (Fig. 3). This set of limits was imposed on each experimental
group to reduce the influence of initial length on reabsorptive half¬
time, and it reduced the number of droplets in the desoxycholate group
from 47 to 33 in 12 animals and in the amphotericin B group from 39 to

23 in 11 animals.

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^eabsorpUVe Half-time (seconds)

Figure Z Correlation of initial droplet I cmfh and
reabsorpti/e half- rime in sil me con¬
trol droplets

(an measured droplets included}

Jfc

12

8

4

0

# • ••

• •

#

#

• •

• •

• #
#

# ®

4 8 11 1b 20

Initial Length (Units)

Coefficient of Correlation:

r= 0.4700, p< o.oi

one hnsi tsiqcr^b ifcitinj 10 /K>it£i9YwD

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a ~f

{fedbscrjjtr/c Half-fin ic (seconds)

17

fom re 3 Correlation of initial droplet length <3nd

feahsorptlvz v l saline control

droplefs

(only droplets with, initial Icrflfh between <j
3rd ZD units included?)

Zo

Vd

11

8

4

4 8 iz I6> 20

Initial length (Units)

Coefficient of Correlation:

r= 0.2755, p>o.i

i Him c s',c«nb ylno)

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18

2. Effect of the droplet composition on reabsorptive half-times.

When t%s were calculated in each group within the aforementioned

limits, the saline control t% was 10.90 ± 0.56 seconds (mean + S.E.),
the desoxycholate mixture was 9.44 ± 0.60 seconds and the amphotericin B
was 7.54 ± 0.61 seconds (Table 2). A comparison of the calculated mean
reabsorptive half-times demonstrates that with amphotericin B in the drop¬
let, the t^ was significantly shorter than with saline (p<0.001) or
desoxycholate (p<0.02) in the droplets. It also appeared that the
desoxycholate mixture alone may have caused more rapid reabsorption
than the saline, although the difference was of borderline significance
(p<0.05). Nevertheless, amphotericin B definitely appears to have an
effect on tubular reabsorption when this agent is in contact with the
luminal membrane of the rat proximal tubule .

3. Calculation of net water flux.

When measured with the stage micrometer, the average internal
radius of the oil filled tubule was 17.8 ± 0.3 y. Net water fluxes for

each experimental group were calculated, based on this average value,

-432 -4

and were found to be: -5.67 x 10 mm /mm /sec. for saline, -6.54 x 10

3 2 “4 3 2

mm /mm /sec. for the desoxycholate, and -8.19 x 10 mm /mm /sec. for

the amphotericin B group (Table 3). The negative values indicate net

water efflux.

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Table 2

Effect of amphotericin & and JesoKycho/afe on
reabsorptive half time.

Sal/he.

Desoxycho/ate

AmpnettridnB

Number

cfAnur&is

Number
of Droplets

Reshsorptiuts
H&it-t/me (Seconds)*

14

33

10.10 -t 0.5b

u

S3

9.44+ 0-&0

11

23

7- 54 - O. <t>!

Significance of differences:

^Saline- Amphotericin 3, p ri 0.00/

Saline - Ocsoxychoiate , p <^0.05
Desoxychofa fc -Ampho fericin B, p<Co.oz

* valued nepnesenT means - standard errors

20

Tabic 3 Effect of a mphoicndn Band descry cholafe

on net waster - Flux

't/et Mkrfhx

(Seconds) Imrrf'Jmrnysec)*

to. f o

Saline
Desoxycfoo/atc
Amphotericin B —

- 5. 67 X /O"*

-6. 5* * so

-4

-8./9X /O

'negative values indicate net flux

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21

DISCUSSION

Reabsorption of sodium in the proximal tubule.

The experiments were designed to evaluate the influence of per¬
meability characteristics of the plasma membrane of the proximal tubular
epithelial cell on net sodium efflux. In particular, the role of the
luminal membrane as a rate limiting factor in sodium entry into the cell
was studied. Amphotericin B was used in this in vivo preparation to
effect an increase in the permeability of the luminal membrane, since
recent studies have demonstrated that when this agent is applied to the
mucosal surface of many epithelial structures in in vitro preparations,
there is an increase in permeability of the affected membrane.

The importance of such cellular factors as membrane permeability
in the regulation of net sodium reabsorption is best understood when
studied in relationship to other factors which are known or thought to
influence sodium reabsorption in the proximal segment of the nephron and
in the kidney as a whole.

From the early work of pioneers in micropuncture technique. Walker
and his associates (7), it can be seen that approximately two-thirds of
the glomerular filtrate is absorbed along the course of the mammalian
proximal convolution. This process occurs while there are no osmotic
or sodium concentration gradients between the tubular fluid and plasma.
These basic findings have been repeated by several investigators and
are now accepted principles upon which the understanding of renal
function is based (8,9,10). The mode of transfer of this large per¬
centage of glomerular filtrate is widely believed to be based on the
active transport of ionic sodium with the water following passively
along the osmotic gradient produced by this solute shift. From

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22

electrochemical determinations across the proximal tubule, it has been
shown that sodium is actively pumped out of the tubule since it can be
demonstrated that sodium passes from lumen to peritubular fluid against
an electrochemical gradient even when a poorly reabsorbable solute re¬
mains in the tubular lumen as during mannitol diuresis (8,10,11). It
is further known that renal sodium reabsorption and oxygen consumption
are linearly related, so that each mole of oxygen used is associated
with the reabsorption of 20-30 mEq of sodium, thus establishing the
energy dependent nature of sodium extrusion (12,13).

For purposes of highlighting reabsorptive mechanisms, different
experimental conditions can be imposed which vary the amount of sodium
that is reabsorbed by the proximal tubule and ultimately excreted in
the urine. It is important to understand what factors have been delin¬
eated which mediate this control of proximal tubular fluid reabsorption.
The first variable to be investigated is the glomerular filtration rate,
a parameter which controls the amount of solute presented to the prox¬
imal tubule. Lindheimer, et a^L. (14) demonstrated that an increase in
glomerular filtration, independent of a change in extracellular volume,
was related to only minimal increases in sodium excretion. This finding
is consistent with the concept of glomerulotubular balance established
by Homer Smith (15), wherein the nephron maintains a balance between
glomerular filtration and tubular reabsorption. It was felt that the
proximal tubule compensated for an increased sodium load by increasing
its absolute reabsorption so that at any point along the tubule, the
percentage of glomerular filtrate remaining was unchanged from the con¬
trol situation (16). But other studies have shown that this proximal
tubular compensation is not perfect under all conditions (4,17), and
that glomerulotubular balance is not an unvarying, inherent property

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23

of the nephron. This compensatory mechanism seems totally absent when
the proximal tubule is perfused in situ by a pump, bypassing the glomer¬
ulus (18). In this case absolute sodium reabsorption remains the same
over wide variations in perfusion. These studies tend to indicate that
glomerulotubular balance does exist under non-diuretic conditions in the
intact nephron, but alterations in extracellular volume or renal blood
flow and changes in filtration independent of the peritubular environment
can upset their relationship.

The second factor classically considered to control sodium reabsorp¬
tion is the effect of adrenal mineralocorticoids . If the composition of
the urine is examined before and after administration of aldosterone, it
becomes apparent that the changes occurring in electrolyte excretion are
primarily a result of increased exchange of sodium for potassium in the
distal tubule (19). It has been shown, however, that aldosterone also
exerts some effect on proximal tubular function as, in adrenalectomized
animals, proximal tubular reabsorption is decreased from control levels,
but returns to normal after the animals receive aldosterone (20,21).

Recently, micropuncture methods have been extensively used to
elucidate other factors involved in the control of sodium reabsorption.
Tubular volume and geometry have been investigated in several situations.
Gertz, et_ al. (4) presented evidence that total transtubular reabsorp¬
tion is a function of the square of the tubular radius. The influence
of tubular geometry was further reinforced by the studies of Rector and

associates (22) who found proximal tubular reabsorption proportional to

2

cross-sectional area (it r ) under stopped flow conditions. Schnermann
and his colleagues (23) demonstrated that even under conditions of
water diuresis, the reabsorptive rate is linearly related to tubular
volume, which is also a function of the square of the tubular radius.

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24

Lewy and Windhager (24), however, found that by increasing the peritubular
capillary pressure via partial constriction of the renal vein, they pro¬
duced a decrease in sodium reabsorption which was not accompanied by a
proportional decrease in the square of the tubular radius. These experi¬
ments indicate that tubular volume is not the sole determinant of
reabsorptive rate, and that under certain experimental conditions, such
as those dealing with alterations in renal hemodynamics, there may be
changes in reabsorption independent of variations in tubular geometry.

In the studies of Earley and his associates (25,26,27), it was
shown that sodium excretion is increased by induced renal vasodilatation,
varies in the same direction as renal perfusion pressure, and is decreased
by infusion of hyperoncotic albumin. When the factor of peritubular
capillary hydrostatic pressure was evaluated in micropuncture studies,
it was shown that increased pressure was associated with decreased fluid
reabsorption (24). Experiments in which hypertension was acutely induced
by carotid artery ligation and bilateral vagotomy, demonstrated that the
elevation in systemic blood pressure was associated with increased urine
flow and sodium excretion (28). Micropuncture techniques demonstrated
definitely decreased fluid reabsorption in the proximal tubules assoc¬
iated with increased hydrostatic pressure within the tubular lumen. The
authors assumed that there was an equivalent increase in peritubular
capillary pressure which was felt to mediate the reduced reabsorption
from the tubule.

The factor of colloid oncotic pressure was investigated by per¬
fusing peritubular capillaries with solutions of varying colloid concen¬
trations and performing free-flow and stationary perfusion micropuncture
on the accompanying proximal tubules (29). With this technique, Spitzer
and Windhager demonstrated that fluid reabsorption from the proximal

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25

tubule varies directly with peritubular oncotic pressure. These studies
all tend to lend credence to Earley’s stress on peritubular determinants
of sodium reabsorption and excretion, but the relative importance of
hydrostatic pressure and colloid oncotic pressure to one another in the
control of reabsorption is still subject to question. The mode of
action of these influences on reabsorption is also hypothetical at this
point, although both Koch, et al. (28) and Windhager's group (24,29)
have suggested increased back diffusion of sodium across the peritubular
membrane or an indirect inhibition of active sodium transport as possible
mechanisms .

Another area involving control of sodium reabsorption which has
aroused some interest is the possibility of a circulating natriuretic
hormone produced during volume expansion. This notion was first sug¬
gested by deWardener, et al. (30) on the basis of experiments in which
dogs, treated with large amounts of mineralocorticoids and vasopressin,
responded to saline infusion with a rise in urinary sodium excretion
despite the deliberate depression of glomerular filtration rate. Whole
sera and fractions of sera from such volume expanded animals were tested
on rats with micropuncture techniques, and initially Rector, et_ al_ . (31)
proposed that a hormone had been found which would inhibit sodium reabsorp¬
tion when infused intravenously into a non-volume-expanded test animal or
placed directly in the proximal tubular lumen. Other investigators using
similar methods, however, have failed to duplicate these findings (32,33).
The controversy over whether such a hormone exists or whether the effects
of volume expansion on sodium reabsorption are entirely mediated by peri¬
tubular factors is still unsettled.

Other possible mechanisms of control of sodium reabsorption have
been suggested. An attractive hypothesis defended by convincing experi-

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26

ments was set forth by Horst er and Thurau (34). They demonstrated that
in addition to mechanisms which regulate sodium reabsorption in different
segments of the nephron, alterations in the function of different portions
of the population of cortical nephrons could also exert a controlling
influence. In this analysis, they distinguished two separate populations
of cortical nephrons, superficial and jux tame dullary. By calculating
single nephron glomerular filtration rates, they found that on a low
sodium diet, individual superficial tubules had filtration rates that
were less than one-half those of jux tame dull ary tubules. However, when
rats were maintained on extremely high sodium diets with saline for
drinking water, the superficial, short-looped nephrons filtered fluid
at greater than twice the rate of the juxtamedullary , long-looped group
although there was only a minimal increase in the total kidney glomerular
filtration rate. It was felt that by altering renal hemodynamics
(possibly via the renin-angiotensin system), the organism was able to
cope with states demanding sodium excretion or sodium retention by
adjusting the distribution of flow to either the sodium wasting (super¬
ficial) or sodium retaining (juxtamedullary) nephrons. Therefore, with¬
out altering the inherent reabsorptive capacities of the proximal tubules,
total kidney tubular reabsorption could be varied by the distribution of
renal blood flow. This concept is supported by the finding that, in
clinical states associated with sodium and fluid retention, superficial
cortical blood flow is depressed to a greater extent than is flow to the
rest of the cortex and medulla (35).

The description of the events of sodium and water reabsorption at
the cellular level has been approached with many different techniques by
the morphologist, the histochemist , the electrophysiologist and the
enzyme biochemist. Recent reviews have incorporated much of this data

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27

into a working model which has been the basis for the explanation of
many experimental manipulations (36,37,38). They describe a multi-
compartmental system in which the process of sodium reabsorption occurs
as follows. Sodium ions cross the relatively permeable luminal membrane
into the epithelial cell down an electrochemical gradient with water
passively following out of the lumen by osmotic flow. These cations are
then actively extruded into the basal labyrinth and the long intercel¬
lular channels. The latter are structures which are closed at the luminal
end in tight junctions and widen to empty into the peritubular space.

It has been shown that the bulk of demonstrable intracellular sodium and
an enzyme system, sodium-potassium activated adenosine triphosphatase,
are both associated with the membranes forming the structures described
above (39,40). This enzyme is one which has long been implicated in
descriptions of the postulated sodium pump in many actively transporting
epithelia, including the renal proximal tubule (41). More recent evidence
suggests, however, that this enzyme may not be the only one involved in
extrusion of intracellular sodium, but that another enzyme may be more
effective in regulating cell volume by expelling sodium accompanied by
chloride ions, and not requiring the exchange of potassium for activation
(42). Once the sodium ions have been actively transported out of the
epithelial cell, Diamond (43) postulates that the ions set up "standing
osmotic gradients" which cause water to flow out of the cells into the
basal labyrinth and down the intercellular channels to accompany the
sodium and return the solutions to isotonicity by the time they reach
the peritubular environment. It is felt that it is this constant
extrusion of solute and water toward the peritubular space which may
be sensitive to the physical forces of hydrostatic pressure and colloid
oncotic pressure, discussed above.

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28

From this brief review it can be seen that many factors are known
to exert some control over net sodium efflux in the proximal tubule.

The relationships of these factors of filtration rate, hormones, tubular
geometry, peritubular forces and functional heterogeneity of the nephron
population to one another and to the minute-to-minute control of proximal
tubular reabsorption are not well comprehended. Other aspects, involved
in control of the active and passive phases of sodium transport at the
cellular level, are appreciated, but not well understood. The studies
described in this presentation were performed in an attempt to evaluate
the nature and influence of an aspect which has been largely neglected
thusfar, the permeability of the luminal membrane. The experiments were
designed to examine what effect alterations in this barrier would have
on sodium transport. This change in permeability was mediated by ampho¬
tericin B, an agent which has been shown to be effective in increasing
plasma membrane permeability in several in_ vitro systems.

The effect of amphotericin B.

Amphotericin B is a polyene antibiotic which was originally pro¬
duced by the actinomycete , Streptomyces nodosus . It is the drug of
choice for treatment of any deep-seated mycoses, but it has an unfor¬
tunately large number of toxicities, including reasonably frequent renal
damage (44). This renal toxicity has been extensively investigated both
clinically and experimentally, and it is felt that most of the damage is
attributable to a vasoconstrictive effect of the antibiotic causing
depression of perfusion and subsequent lowered glomerular filtration
rate (45,46). More recently, the pathological picture of nephrocalcinosis
and the occurrence of hypokalemia and hyposthenuria occasionally preceeding
azotemia was investigated by McCurdy, et_ al. (47). They found that patients

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29

being treated with amphotericin B frequently demonstrated the clinical
findings of distal renal tubular acidosis.

In the experiments described in this presentation, the dose of
amphotericin B used was based on the work done with the toad urinary
bladder by Lichtenstein and Leaf (48), who found maximal effects of
amphotericin B at 13 yg/ml. In an attempt to guarantee an effect in
the perfused, intact, renal proximal tubule, the initial in vivo dose
was increased to 50 yg/ml, a level which did produce some effect. In
their study, Lichtenstein and Leaf state that sodium desoxycholate , the
solubilizing agent for amphotericin B, produced no significant effect
in their preparation. It was felt that this agent should also be tested
in a mammalian system. Consequently, following experiments on the first
group of rats used both for learning of technique and acquiring control
data, the effects of intratubular injections of two experimental sub¬
stances, amphotericin B and desoxycholate, were evaluated.

The action of amphotericin B was assessed under stop-flow conditions

to diminish the influence of possible systemic effects of this agent.

Therefore, the changes occurring with intratubular application of this

drug can probably be attributed to its local activity. From Table 2,

it can be seen that when amphotericin B was added to the tubular lumen,

net transport increased as evidence by a change in t^ from 10.90 to 7.54

seconds, a significant decrease of greater than 30%. When expressed as

-4 3 2

net water flux, an increase in magnitude from -5.67 x 10 mm /mm per

-4 3 2

second to -8.19 x 10 mm /mm per second was demonstrated. There is
also an apparent decrease in t% from control values associated with
desoxycholate, although amphotericin B produced a further significant
decrease in t^ of approximately 20% from the desoxycholate value. The
difference between the saline control and desoxycholate may, in fact,
be influenced by the improvement in technical ability of the novice

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30

investigator, and its significance is therefore subject to question. In
summary, there is evidence that by the action of amphotericin B on the
luminal membrane of the proximal tubular epithelium, this drug increased
sodium and fluid reabsorption in an isolated segment of mammalian proximal
tubule .

The results in this mammalian system were duplicated and elaborated
upon in experimentation with the proximal tubule of Necturus (49). Stroup
and Kashgarian have found that at concentrations of both 10 yg/ml and 50
yg/ml, amphotericin B reduced the t% to approximately 50% of control
values. It was also found that solute influx into an intraluminal,
isotonic raffinose droplet was significantly increased by amphotericin B.
Finally, in measuring equilibrium concentrations, they found that the
concentration of sodium was unchanged but that of potassium was distinctly
increased. They interpreted this data as an effect of amphotericin B
causing increased passive permeability and depolarization of the luminal
membrane which was associated with increased net transport of solute and
water .

The only other work with this agent in altering membrane permeability
has been in in vitro systems, particularly the toad bladder. Lichtenstein
and Leaf (48) originally used amphotericin B with vasopressin to demon¬
strate the double series permeability barrier originally postulated by
Andersen and Ussing (50) in the toad skin. The membrane, according to
this concept, consists of an outer homogeneous diffusion barrier with low
permeability to most hydrophilic solutes and a deeper porous barrier which
resists bulk water flow, the latter being particularly sensitive to ampho¬
tericin B in Lichtenstein and Leaf’s experiments. Their results were
challenged by Mendoza, et_ al_. (51), using similar techniques, who found
that the effect of amphotericin B on the toad bladder was not limited to

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31

only one of the barriers. They cautioned that the assumption that ampho¬
tericin B acts only at a single site was not proven, and that the actions
of other agents should not be interpreted on this basis. Finn (52), also
using the toad bladder, determined that amphotericin B was active at both
serosal and mucosal membranes, causing increased passive movement of sodium
ions across the membrane facing the medium to which amphotericin B had been
added. Singer, et al. (53) have evaluated many of the variables affecting
the toad bladder preparation including season of the year, time after dis¬
section, initial short-circuit current and initial potential difference
across the membrane. They found that under certain conditions, the orig¬
inal observations of Lichtenstein and Leaf (48) could be consistently
reproduced and reaffirmed the double barrier membrane hypothesis. The
effects of amphotericin B on the toad bladder were elegantly studied by
Saladino, et al. (54), who correlated the electrophysiological and ultra-
structural changes in mucosal cells during exposure to this agent over
various periods of time. They found that amphotericin B reduced the
capacity of apical plasma membranes to maintain intracellular gradients.
After a few hours of incubation with the antibiotic, structural and
functional disintegration occurred, with intracellular swelling and mito¬
chondrial matrix condensation. Ultimately, there was necrosis with
swelling of intracellular ground substance and disruption of many organ¬
elles. In the early phases, at two and eight minutes, before demonstrable
morphological cell disruption could be demonstrated, there was a persistent
increase in short-circuit current implying increased transport by a still
competent, unsaturated sodium pump. This parameter fell in concordance
with the demonstrated breakdown of cellular integrity during more prolonged
incubations .

Other in vitro systems have been examined with amphotericin B.

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32

Lippe and Giordana (55) exposed the small and large intestines of the
tortoise to various concentrations of amphotericin B and found that at
the same dosage of drug, permeability to thiourea was increased far more
in the large intestine. They also noted that permeability changes seemed
to affect the transport of smaller molecules much more than that of mole¬
cules which were the size of creatinine or larger, and that there was no
change in the permeability to lipid soluble molecules. Steinmetz and
Lawson (56) found that in the turtle bladder, amphotericin B caused a
defect in urinary acidification due to increased passive permeability of
the luminal membrane resulting in increased back diffusion of hydrogen
ion. It was also shown that there were larger increases in permeability
to potassium than to sodium and chloride, and the authors suggested that
this model might be analogous to the clinically described renal tubular
acidosis found in some patients being treated with amphotericin B (47).

These in_ vitro studies generally tend to support a consistent action of
amphotericin B of increasing the passive permeability of most membranes
facing media containing this drug, rather than any direct influence on
active transport in the system being examined.

The mode of action of this agent in altering membrane permeability
has been studied for several years. The susceptibility of fungi, but not
bacteria, to the polyene type of antibiotic suggested that some difference
in the composition of the plasma membrane was the basis of differential
sensitivity to these drugs. Working with nystatin, a polyene with a mode
of action similar to that of amphotericin B, and fungal protoplasts,

Lampen, et_ al . (57) suggested that antibiotic binding by the plasma membrane
was the critical event in cell damage and that the binding site contained
sterols, types of lipids which are not found in bacteria. Furthermore,
studies comparing mammalian erythrocytes with fungal protoplasts demon¬
strated that, at low concentrations of polyenes, there was a rapid lysis

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33

of both types of cells, indicating that mammalian membranes, too, were
susceptible to these drugs and that the induced changes caused greatly
increased membrane permeability (58). Using the physical chemical con¬
cept of "lipid monolayer penetration", Demel and his associates (59)
found that the sterol moiety, rather than the phospholipid component, of
plasma membranes was most sensitive to the amphotericin B and proposed
that the mode of action was one of "reorientation" of the sterols in
membranes leading to increased permeability and leakage of intracellular
components. More recent work has indicated that the maximal effect of
amphotericin B on artificial phospholipid-cholesterol structures was
found in certain combinations of phospholipid and sterol, rather than
in pure cholesterol monolayers (60,61). These findings, regarding both
real plasma membranes and lipid analogues, indicate that some sort of
binding of polyene to sterol occurs, leading to a reorientation of the
sterol molecules, resulting in increased permeability of the involved
membrane. The binding aspect of this concept is further supported by
the finding of Mendoza, et al. (51) that ergosterol inhibits the action
of amphotericin B in the toad bladder preparation, presumably by com¬
peting with membrane binding sites for the free antibiotic.

In light of the findings of the many workers who used amphotericin
B in in vitro or chemical systems, some explanation can be put forth for
the increased reabsorption in the mammalian proximal tubule which has
been exposed to this agent. It appears that amphotericin B, from the
intratubular tubular perfusate bathing the luminal membrane , interacted
with this mammalian, sterol-containing membrane for a short period of
time and caused increased permeability without intracellular disruption.
With this relative barrier to solute penetration somewhat attenuated,
increased ionic sodium and water crossed into the cell. Since larger

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:

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34

amounts of sodium were presented to the sodium pump, this mechanism was
able to move the sodium out of the cell at a rate which was more rapid
than normal. Whether this altered capacity for ionic transport was due
to lack of substrate saturation under control conditions or, possible,
to increased ability of an already saturated pump, is subject to question.
The results of Stroup and Kashgarian (49) of unchanged equilibrium con¬
centration of intratubular sodium during amphotericin B exposure,
indicate that active transport is unchanged in the amphibian kidney.
Furthermore, the findings of Sharp and his associates (62) in the toad
bladder, demonstrate that the increased active transport of sodium due
to incubation of the tissue in aldosterone is augmented by treatment
with amphotericin B. This finding can be interpreted as an indication
that a more potent active transport system can be even further respon¬
sive to an increase in substrate level, if that level remains below
the saturation limit of the pump.

The experimental results and this formulation suggest that crossing
the luminal membrane is a rate limiting step in net sodium outflux.
Therefore, altering the permeability characteristics of this barrier
may be an important mechanism in regulating sodium movement across the
proximal tubule, independent of filtration rate, hormones, physical
factors and other postulated influences on sodium transport. The practical
importance of the ability to manipulate this barrier is that it should be
considered a potentially dynamic structure, possibly capable of both
response to and control of altered conditions of solute transport, rather
than a non-variable portal for delivery solute to the pump.

The Split-Droplet Method

The method used in these experiments was the split-droplet technique,

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35

originally developed by Gertz (5). It is an attempt to measure the in¬
trinsic reabsorptive capacities of the proximal and distal tubules,
independent of the effect of filtration rate, and it also affords an
opportunity to manipulate the composition of the perfusate in order to
locally, rather than systemically , affect the reabsorptive apparatus.

In many studies where both free-flow and stop-flow micropuncture techniques
were employed under the same experimental conditions, there has been
excellent correspondence between the results of the two methods (4,6,24,29).
Several investigators, however, have recently questioned the reliability
of this method, and, therefore, attempts were made to control many of
the variables in the experimental technique. The initial droplet length
was standardized? the oil block was adequate to prevent flow but not exces¬
sively long, and observer bias was limited by the measurement of coded
photographs and the statistical regression approach to calculation.

The considerations to be described are included to explain the reasons
why standardization of these variables may be crucial to the reproduc¬
ibility and reliability of this method.

In the presentation of the results of this experimentation, it was
indicated that certain limits were set on the initial length of the drop¬
let. The rationale behind such setting of limits is, in part, based on
the realities of droplet geometry. The concept of considering the inter-
meniscal length to be proportional to volume is based on the assumption
that the droplet shape approaches a regular cylinder with plane bases.

From the pictures of the reabsorption sequence (Fig. 1), it is obvious
that this is an oversimplification. In the photographs, the menisci
appear to approximate semicircles; these would be equivalent to hemispheres
of oil in three dimensions since the radius of the tubule is felt to remain
constant. An expression for the actual volume of the droplet can be

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36

derived based upon this description of the shape of the meniscus (Fig. 4).

The actual volume (v) is equal to the volume of a cylinder, with length
equal to the intermeniscal length (1) plus the radii of both hemispheres
(2r), minus the volume occupied by the two hemispheres:

(8) v = Trr^ (1 + 2r) - 2 • 2/3 Trr^ - mr" (L = 2/3r).

It can be readily seen, therefore, that as an approximation of
2

volume, nr 1 becomes a smaller proportion of the actual droplet volume
as 1 decreases, approaching r. Steinhausen (63) originally described
this phenomenon, noting that at initial lengths less than 90 y, a distance
approximately equal to five tubular radii (5r), there was a definite
correlation between initial length and t^. In recent experiments performed
in this laboratory, a lower limit of initial length equal to 4r has been
set for acceptable droplets. This is based on Weinman's (64) finding
that a plot of initial length against t^ disclosed a region from 60 y to
90 y (approximately 4r to 6r) over which there was no significant cor¬
relation between these variables, but, above and below these limits, there
seemed to be an effect of initial droplet length on calculated reabsorption.
If these criteria had been used for the data in these experiments, too
small a sample size would have resulted, and I therefore, compromised
and used approximately 3r as my lower limit (tubular radii equaled 3-3^
units). This value was consistent with the minimal droplet length cri¬
terion of approximately 2%p originally established by Naka j ima , et al.

(65) in canine proximal tubules. It was, however, far short of the 100 y
minimum (approximately 7r) recently used by Bank's group (66).

The upper limit of 20 units is approximately 6r which was consistent
with limits suggested by Weinman (64), and it provided an overall range of
3-6r which produced a control distribution of t^ that was sufficiently
random between the limits so that there was no significant correlation

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37

figure 4 Approximation of actual

droplet Volu/'Vie.

4 - L - ►

j i - L + Zf- - ►!

V =■ trr£ Cu2r)~ 2 • §trr 3 - it r* (u-§r)

i ♦* ^ .

38

with initial length (Fig. 3). Furthermore, even if one accepted the claim
of Levinsky, et al. (67) that in their hands, there is no range within
which is not affected by initial length, my setting of limits within
which there were reasonably similar distributions of initial lengths for
all experimental groups would tend to control for this effect.

There have recently been other criticisms of the split-droplet
technique and attempts to revise it by altering methods of measurement,
calculation and the injection of the intratubular oil block. Many tech¬
niques have been devised for measuring the lengths of droplets which may
not be totally straight. Among these are the projection of colored trans¬
parencies or black and white negatives of photographs of droplets, with the
intermeniscal distances determined by several means. These include the use
of planimeters or mapreaders (28,31), paper cut-outs of the outlines of
tubules with the estimation of the percent decrease in volume based on
weight change (29), and division of the projected droplets into roughly
rectangular midplane, cross sectional areas with the summation of these
areas as the representation of tubular volume (68). Bentzel, et al. (69)
have used formula (8), with direct measurements of intermeniscal length
and tubular diameter to better approximate actual tubular volume.

To calculate t^, all of these methods involve a semilogarithmic
plotting of percent change in the parameter representing volume against
time, with a best-fit line drawn by eye. In the experience in this lab¬
oratory, attempts to draw this line have often been complicated by the
lack of colinearity of all points and decisions were made whether to
neglect the aberrant point (s) or to try to adjust the best-fit line.

For these experiments, in order to give adequate consideration to all
data collected on each droplet , the changes in intermeniscal distance
were calculated into a statistical regression. In this computation, the

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39

slope of each point from the initial point was weighted equally, thus
eliminating another possible source of observer bias.

Most investigators continue to calculate t^g based on some parameter
representative of tubular volume. In their study, Nakajima, et_ al. (65),
concluded that the factor actually being varied in the split-droplet
technique was the reabsorptive surface area exposed to the droplet. In
recalculating their data, they found no effect of initial length on t?g,
if the regression of the total length of tubule exposed to the droplet
(including the area behind each meniscus) was calculated. Although this
was not Gertz's (4,5) original formulation, it may well be that their
rationale, which seemed to evolve from a mathematical manipulation per¬
formed to eliminate the influence of initial length on t^g, is correct.

Some attention has recently been paid to the nature of the oil block.
Levinsky, et_ al_ . (67) found that the length of the distal oil block had no
effect on control studies, but, after volume expansion, the large oil block
was associated with much less of an increase in t^g than the smaller block.
Nakajima, et_ al_. (65) found a difference in t^g when the composition of the
oil was. altered and also raised the possibility that there were different
frictional resistances to movement affecting the proximal and distal oil
blocks. Finally, they suggested that there might be adverse effects of
the oil on reabsorption. This criticism can be refuted in part, by the
correlation of ultrastructural and physiological observations on the effect
of oil on the reabsorptive epithelium performed by Wiederholt and his
associates (70). They demonstrated that contact with castor oil and
tubular dilatation with the oil block do not damage the epithelium to
any measurable degree, as judged morphologically and functionally, even
after several applications of the technique to the same segment.

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40

It can be seen that there are several possibilities for intro¬
ducing methodological artifacts into the assessment of reabsorption by
the split-droplet technique. I believe, however, that the limitations
placed on this basically reliable method in these experiments have per¬
mitted the generation of data which represent actual physiological
alterations and demonstrate a real effect of amphotericin B on the
mammalian proximal tubule .

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41

SUMMARY

The effect of the intratubular injection of amphotericin B on
proximal tubular reabsorption was examined using the split-droplet
micropuncture method. Under these conditions, reabsorptive half-time
was significantly decreased, indicating an increase in the rate of
proximal tubular reabsorption. This was interpreted as being due to
a more permeable luminal membrane allowing increase passive efflux
of sodium and water. These studies implied that the luminal membrane
was a rate limiting barrier in proximal tubular sodium reabsorption.

YHAMM'J.?

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42

REFERENCES

1. Wirz, H. Druckmessung in Kapillaren und Tubuli der Niere durch

Mikropunkture . Helvet . Physiol . et Pharmacol. Acta , 13: 42, 1955.

2. Steinhausen, M. Eine Methode zur Differenzierung proximaler und

distaler Tubuli der Nierenrinde von Ratten in vivo und ihre
Anwendung zur Bestimung tubularer Stromungsgeschwindigkeiten.
Pflugers Arch. ges. Physiol., 277: 23, 1963.

3. Gottschalk, C. W. and Mylle , M. Micropuncture study of pressures

in proximal tubules and peritubular capillaries of the rat kidney
and their relations to ureteral and renal venous pressure. Am .

J. Physiol. , 185: 430, 1956.

4. Gertz, K. H., Mangos, J. A., Braun, G. and Pagel, H. D. On the

* 9

glomerular tubular balance m the rat kidney. Pflugers Arch.
ges. Physiol. , 285: 360, 1965.

5. Gertz, K. H. Transtubulare Natriumchloridflusse und Permeabilitat

fur Nichtelektrolyte im proximalen und distalen Konvolut der
Rattenniere. Pflugers Arch, ges . Physiol. , 276: 336, 1963.

6. Hayslett , J. P., Kashgar ian , M. and Epstein, F. H. Functional

correlates of compensatory renal hypertrophy. J. Clin. Invest.,

47: 774, 1968.

7. Walker, A. M. , Bott , P. A., Oliver, J. and MacDowell, M. Collection

and analysis of fluid from single nephrons of the mammalian kidney.
Am . J_. Physiol. , 134: 580, 1941.

8. Ullrich, K. J., Schmidt-Nielsen , B., O’Dell, R. , Pehling, G.,

Gottschalk, C. W., Lassiter, W. E. and Mylle, M. Micropuncture
study of proximal and distal tubular fluid in rat kidney. Ain. J_.
Physiol. , 204: 527, 1963.

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.MOi f VS 3 : ■PCS e ._Ioi;^qj

43

9. Clapp, J. R. , Watson, J. F. and Berliner, R. Osmolality, bicar¬
bonate concentration, and water reabsorption in proximal tubule
of the dog nephron. Am. J_. Physiol. , 205: 273, 1963.

10. Windhager, E. E. and Giebisch, G. Micropuncture study of renal

tubular transfer of sodium chloride in the rat. Am. J. Physiol. ,
200: 581, 1961.

11. Kashgarian, M. , Stockle, H. , Gottschalk, C. W. and Ullrich, K. J.

Transtubular electrochemical potentials of sodium and chloride
in proximal and distal renal tubules of rats during antidiuresis
and water diuresis (diabetes insipidus). Pflugers Arch, ges .
Physiol. , 277: 89, 1963.

12. Kill, F. , Aukland, K. and Refsum, H. E. Renal sodium transport

and oxygen consumption. Arn. J. Physiol. , 201: 511, 1961.

13. Deetjen, P. and Kramer, K. Reabsorption and oxygen consumption in

the mammalian kidney. Klin. Wchschnr . , 38: 680, 1960.

14. Lindheimer, M. D., Lalone, R. C. and Levisky, N. G. Evidence that

acute increase in glomerular filtration has little effect on sodium
excretion in the dog unless extracellular volume is expanded.
iL' Clin. Invest . , 46: 256, 1967.

15. Smith, H. W. The Kidney: Structure and Function in Health and

Disease , p. 460, New York, 1951. Oxford University Press.

16. Dirks, J. H. , Cirksena, W. J. and Berliner, R. W. Effects of saline

infusion on sodium reabsorption by proximal tubule of dog. J.

Clin. Invest . , 44: 1160, 1965.

17. Brenner, B. M., Bennet , C. M. and Berliner, R. W. Relationship

between glomerular filtration rate and sodium reabsorption by
proximal tubule of rat kidney. J. Clin. Invest., 47: 1358, 1968.

rrenilri9fi fjtrs . 1 ' (no EdBW , .H > b { .[Q£!"-‘

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44

18. Morgan, T. and Berliner, R. W. In vivo perfusion of proximal

tubules of the rat: Glomerulotubular balance. Am_. J. Physiol. ,

217: 992, 1969.

19. Mills, J. N. , Thomas, S. and Williamson, K. S. Effects of intra¬

venous aldosterone and hydrocortisone on urinary electrolytes
of the recumbent human subject. J. Physiol . , 156: 415, 1961.

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46

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47

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1968.

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