Download DISTRIBUTION OF PEROXISOMES - The Journal of Cell Biology

Published August 1, 1969
DISTRIBUTION OF PEROXISOMES
(MICROBODIES) IN THE NEPHRON OF THE RAT
A Cytochemical Study
MARGARET E . BEARD and ALEX B . NOVIKOFF
From the Department of Pathology, Albert Einstein College of Medicine of Yeshiva University,
Bronx, New York 10461
ABSTRACT
INTRODUCTION
Previous studies of renal peroxisomes (or microbodies) have been either biochemical in nature (1,
2, 5, 6, 9, 10) or electron microscopic (11, 14, 15,
23, 33, 34, 35, 49, 62) . Biochemically, these organelles have been characterized as a distinct
group of intracellular particles that : (a) sediment
in sucrose density gradients at an equilibrium position more dense than that of lysosomes and mitochondria ; (b) behave as partial osmometers ; and
(c) contain a population of distinctive oxidative
enzymes . Electron microscopically, peroxisomes
have been described as membrane-bounded bodies
of moderate electron opacity, approximately 0 .51 .0 s in diameter (49) . When first studied in mouse
kidney, no internal structure was described (49) . In
rat kidney, Ericsson (11) and Maunsbach (35)
have identified a relatively amorphous, slightly
eccentric and slightly electron-opaque "core ."
Trump and Ericsson (62) have also described
tubular elements projecting from renal peroxisomes .
The fine structure of peroxisomes has been more
completely studied in hepatocytes (see 10) . In
these cells, the organelles are round to slightly
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The distribution of peroxisomes (microbodies) in the rat nephron was studied cytochemically,
using glutaraldehyde- or formaldehyde-fixed tissue, by means of a-hydroxy acid oxidase
activity in light microscopy of oxidation of 3 , 3'-diaminobenzidine (DAB) at pH 9 in both
light and electron microscopy .The two cytochemical methods show peroxisomes to be nearly
sperical particles found only in cells of the proximal convoluted tubule . Lysosomes were
identified in the same or parallel sections, with ,Q-glycerophosphate or 5'-cytidylic acid as
substrate . They are found in all cells of the nephron . These cytochemical methods visualize
the two organelles for light microscopy ; they also permit unequivocal differentiation of all
kidney peroxisomes from lysosomes in electron micrographs . Peroxisomes are larger and
more reactive in the cells of the pars descendens (P 3 segment) of the proximal convolution,
located in the outer medulla and medullary rays, than in the cells of the pars convoluta (P 1
and P 2 segments), situated in the cortex . In contrast, lysosomes are much smaller in the P 3
segment and larger and more reactive in the P 1 and P 2 segments . In all cells of the proximal
convolution, peroxisomes tend to be concentrated nearer the base of the cells than do lysosomes . Mitochondria in P 3 cells also show low levels of DAB oxidation at pH 6, in contrast
to those in P 1 and P 2 cells . The possibility is discussed that P 3 cells possess an extramitochondrial means of oxidation in which peroxisome oxidases play an important role .
Published August 1, 1969
elongate and contain a moderately electronopaque nucleoid or core (except in man) . The
nucleoid of rat hepatic peroxisomes consists of a
regular arrangement of stacked rods or tubules,
suggestive of a crystalloid (5, 23, 25, 63) . Hepatic
peroxisomes of other mammals and other vertebrates often show different types of nucleoids .
Urate oxidase has been associated with the nucleoids in rat liver (24, 53, 63) .
Recently, enzymatic staining reactions have enabled visualization of peroxisomes in tissue sections
prepared for light microscopic study (1, 2, 16, 22,
44, 45) and for electron microscopic study (44, 45) .
These methods facilitate identification of these organelles in electron micrographs, and make possible a more adequate description of their distribution and character in various segments of the renal
tubule . Their size and distribution may more
readily be compared with those of other cellular
organelles.
Adult male rats (Holtzman strain), weighing approximately 200 g and maintained on a diet of Purina lab
chow and water ad libitum, were used in these studies .
Both kidneys were excised while the animal was under
light ether or sodium pentobarbital anesthesia . Thin
transsectional slices, less than 3 mm thick, were
placed in either 3 % glutaraldehyde (Fisher Biological
Grade 50'/',, solution) ; Fisher Scientific Company,
Pittsburgh, Pa.) buffered with 0.1 M sodium cacodylate pH 7 .4 (50) for 6 hr at 4 ä C or in neutral 1%
calcium-4% formaldehyde (3, 4) for 12-18 hr at
4 äC . Following fixation, the tissues were rinsed in 0 .1
M cacodylate buffer pH 7 .4, containing 0 .22 M sucrose,
for at least 12 hr prior to use .
Light Microscopy
With the exception of procedure e below, in which
cryostat-cut sections of briefly fixed (67) or unfixed
tissue was used, frozen sections, 10 ß in thickness, were
prepared from formaldehyde-fixed or glutaraldehydefixed tissue with a freezing microtome . These were
incubated as free-floating sections at 37 äC in the
following media :
a . DAB oxidation medium at pH 9.0 of Novikoff and
Goldfischer (44, 45) modified from Graham and
Karnovsky (19) .
10 .0 ml 0.05 M 2-amino-2 methyl-1,3-propanediol buffer pH 9 .4 ;
0 .2 ml 1 % hydrogen peroxide freshly diluted
from 30% solution (Baker) ;
20 mg
3,3'diaminobenzidine tetrahydrochloride (DAB, Sigma) ;
Filter, if necessary, and adjust pH to 9 .0.
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TILE JOURNAL OF (CLL BIOLOGY
°
of Novikoff and
Goldfischer (44, 45) modified from Graham and
Karnovsky (19) .
10 .0 ml 0.05 M sodium acetate-acetic acid buffer,
pH 5 .0 ;
0 .1 ml 0.1% hydrogen peroxide (diluted from
30% solution, Baker) ;
20 mg
DAB ;
1 .0 ml 0 .05 M manganous chloride ;
Filter, if necessary, and adjust pH to 6 .0 .
c . ce-Hydroxy acid oxidase medium of Allen and Beard
(1, 2) .
d. Acid phosphatase medium modified from Gomori
(17), using Sodium-,Q-glycerophosphate (Sigma
Chemical Co ., St. Louis) or Cytidine-5'-monophosphate, CPM (Sigma Chemical Co .) (41, 42) .
e. Alkaline phosphatase medium of Gomori (18) .
f. Nucleoside diphosphatase medium of Novikoff et al .
(43) .
g. Nucleoside triphosphatase medium of Wachstein and
Meisel (66), substituting manganese chloride for
magnesium sulfate .
h . Glucose-6 phosphatase medium of Wachstein and
Meisel (65) .
i. NADH, tetrazolium reductase medium of Novikoff
et al . (47), using 2,2', 5,5'-tetrap-nitrophenyl3,3'-3,3'-dimethoxy(4,4'-diphenylene) ditetrazolium chloride (TNBT), Sigma .
j. Succinate-tetrazolium reductase medium modified from
Nachlas et al . (38) .
Following incubation, all sections were rinsed in
distilled water. In media d through h, the sections
were treated with dilute ammonium sulfide solution
for visualizing the sites of reaction product . All sections were mounted in glycerogel .
DAB oxidation medium at pH 6.0
Electron Microscopy
25-micra sections of selected areas of either cortex
or outer medulla (see Fig. 1) of glutaraldehyde-fixed
tissue were obtained with the Smith-Farquhar
(Sorvall TC-2) tissue sectioner . Free-floating sections
were incubated in reaction mixtures, as outlined
above, but with the addition of 0 .22 M sucrose, for
reaction sites of acid phosphatase, a-hydroxy acid
oxidase, and DAB oxidation at pH 9 and pH 6.
Following cytochemical incubation, the tissue was
rinsed in 0.1 M cacodylate buffer pH 7 .4, containing
0 .22 M sucrose, and postfixed in osmium tetroxide
(36), generally for 1 hr at room temperature . The
tissue was dehydrated by quick changes in graded
alcohols and propylene oxide and was embedded in
Epon 812 modified from Luft (29) . Thin sections were
cut with a Porter-Blum (Sorvall MT 1) ultramicrotome equipped with a diamond knife and were placed
on 300-mesh uncoated copper grids . They were examined, either unstained or stained with lead (48), on
VOLUME 42, 1969
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MATERIALS AND METHODS
b.
Published August 1, 1969
sections incubated in media lacking substrate or in
reaction mixtures containing inhibitors of enzyme
activity, in medium b 0 .01 M 3-amino-1,2,4,-triazole
(K & K Laboratories, Plainview, N.Y .) for inhibiting
catalase in microbodies ; in medium c, 0 .01 M potassium cyanide for inhibiting mitochondrial oxidation
of DAB .
RESULTS
Fig . I indicates the four regions of the male rat
kidney and the distribution of the various segments
of the nephron in these regions . Three segments of
the proximal convoluted tubule, P1, P2, and P 3 ,
are identified in accordance with the descriptions
of Ericsson (11) and Maunsbach (35) .
PAPILLA
INNER MEDULLA
OUTER MEDULLA
CORTEX
TL
Light Microscopy
an RCA 3-H electron microscope operated at 100 kv
or, occasionally, at 50 kv .
Control sections were always included in both light
and electron microscopic studies . These consisted of
The results of the light microscopic cytochemical
staining reactions found in selected parts of the
male rat nephron are summarized in Table I .
Acid phosphatase-rich lysosomes are present in
all cells of the nephron (Figs . 2 and 4) . In cells of P1
and P2 segments, they are abundant, large, highly
active, and concentrated in the base of the cells
(Fig . 4) . In P 3 cells, they are fewer in number and
smaller than in Pl and P 2 cells ; they are generally
more apically situated (Fig . 4) .
Peroxisomes show the same distribution whether
TABLE I
Evaluation of Light Microscopic Cytochemical Staining
Proximal convoluted tubule
Lysosomes
Acid Pase
Microbodies
a-OH ac . ox .
DAB Oxid . pH 9
Mitochondria
Succ-TNBT-Reduc .
NADH2-TNBT-Reduc .
DAB Ox . pH 6
Endoplasmic Reticulum
G-6-Pase
NDPase
Plasma Membrane (Brush border)
ATPase
Alk Pase
Ascending limb
loop of Henle
P,, P,
P,
Large
++++
Smaller
++
+++
Small
++
Larger
++++
++++
Very small
+
++ .
+++
+++
++
++
+
++++
++++
+++
(0)
(0)
+++
0
0
++
++
+
0
++++
o
0
0
++++
0, no reaction ; +, barely detectable reaction ; ++, light reaction ; +++, moderate
reaction ; ++++, intense reaction . Parentheses indicate an equivocal result .
M. E . BEARD AND A .
B.
NovIEOFE
Peroxisome Distribution
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Diagrammatic transsection of rat kidney
showing the distribution of segments P1, P2 , and P3
of the proximal convoluted tubule . B, Bowman's
capsule ; ThL, thin limb of Henle's loop ; TL, thick
limb of Henle's loop ; D, distal convoluted tubule ;
C, collecting duct .
FIGURE 1
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FIGURE 2 10-µ frozen section of glutaraldehyde-fixed kidney, incubated in CMP medium for 10 min
at 37 ° C . Reaction product is most concentrated in segments Pi and P 2 . At higher magnification, the
reaction product is seen in lysosomes (see Fig . 4) . P3 cells have smaller and less reactive lysosomes
than do PI and P2 cells (see Fig . 4) . At this magnification, the small and sparsely distributed lysosomes
in other parts of the nephron are not visible . X 25 .
FIGURE 3 10-µ frozen section of glutaraldehyde-fixed kidney incubated in DAB medium pH 9 for 30
min at 37 °C. Reaction product is concentrated in P3 cells . At higher magnification (see Figs . 5 and 8)
the reaction product is seen localized in peroxisomes . The staining in P1 and P2 cells is markedly lighter .
X 25 .
Published August 1, 1969
FIGURE 5 10-µ frozen section of calcium formaldehyde-fixed kidney incubated in o .-hydroxyvalerate
medium for 20 min at 37 °C . The peroxisomes in P3 cells are more numerous and darker than in PI and
P2 cells . No reaction product is visible in the glomerulus (G) or other regions of the nephron . X 130 .
visualized by DAB oxidation at pH 9 (Figs . 3, 6,
chondria . At higher magnification (not illus-
and 8) or by a-hydroxy acid oxidase activity
(Fig . 5) . Viewed by electron microscopy as well as
trated), the mitochondria of P I and P 2 appear as
light microscopy, peroxisomes are restricted to
basal plasma membrane . Mitochondria of P 3 cells
cells of the proximal convolution . In contrast to
are more spherical and are randomly distributed
the situation for lysosomes, peroxisomes are smaller
throughout the cytoplasm . These observations are
and less abundant in PI and P 2 cells than in P 3
strengthened by electron microscopy .
elongate structures oriented perpendicularly to the
cells . The peroxisomes are more basally situated
It should be noted that the strongest mitochon-
than most lysosomes in cells of all three segments
dria) reaction by all procedures is given in cells of
of the proximal convolution . To date, our attempts
the thick limbs of Henle's loop (Fig . 7) .
to demonstrate peroxisomes at the electron microscopic level by virtue of their a-hydroxy acid oxidase, with use of even the best tetrazolium salt for
this purpose (52), have been unsuccessful .
Electron Microscopy
Differences in cell ultrastructure in different
segments of the proximal convolution have been
DAB oxidation at pH 6 visualized mitochondria
described (11-15, 20, 30-35) . The morphological
(Fig . 7) in a pattern comparable to that demon-
differences between cells of segment P I , a short
segment immediately following Bowman's capsule,
strated by NADH2-tetrazolium reductase activity
and succinate-tetrazolium reductase activity (not
illustrated) . The PI and P2 cells stain more strongly
than P 3 cells in all media which demonstrate mito-
and P 2 cells are much less pronounced than those
between both P I and P 2 cells and cells of P 3 . This
is reflected in the light microscopy described above,
M. E. BEARD AND A . B . NovIKOFF
Perorisome Distribution
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FIGURE 4 10-µ frozen section of glutaraldehyde-fixed kidney incubated in CMP medium for 10 min at
37° C . The lysosomes are darker in PI and P 2 cells than in P3 cells . The lightly stained lysosomes in cells
of the loop of Henle (arrow) are barely evident . X 130.
Published August 1, 1969
min at
frozen section of glutaraldehyde-fixed kidney incubated in DAB medium pH 9 for
P3 portions are stained more deeply than PI and P2 portions . X 130.
10-p
37°C .
30
FIGURE 7 10-p frozen section of glutaraldehyde-fixed kidney incubated in DAB medium pH 6 for 60
min at 37 ° C. Reaction product, due to mitochondrial reactivity, is more evident in PI and P2 portions
of the nephron than in P3 portions . Mitochondrial staining is most intense in cells of the thick limbs of
Henle's loop (arrow) . This is even more evident in sections incubated for shorter periods . X 130.
where no distinction between P I and P 2 cells could
be made .
In PI and P 2 cells (Figs . 11 and 13), the brush
border is of moderate length, the plasma membrane is highly infolded, and many of the mitochondria are elongate, are oriented perpendicularly to the base of the cells, and lie between the
cytomembranes . Endoplasmic reticulum is sparse .
In P 3 cells (Figs . 10, 12, and 14), the microvilli
of the brush border are longer. Larger amounts of
endoplasmic reticulum than in P I and P 2 cells are
seen . Most mitochondria, but not all, are oval
rather than elongate . They are distributed in all
but the most apical portions of the cytoplasm
without evident orientation . The basal infoldings
of the plasma membrane are neither as evident nor
as deep as in the P I , P2 cells .
In both P I and P 2 cells and P 3 cells there are
some cytoplasmic dense bodies for which the
identification as either peroxisome or lysosome is
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THE JOURNAL OF CELL BIOLOGY . VOLUME 42,
difficult to make in electron micrographs . However, by incubating sections for acid phosphatase
activity, or for DAB oxidation at pH 9, or both,
their identification is readily made . Acid phosphatase reaction product is coarsely granular, irregular, and highly electron opaque ; DAB oxidataion product is less electron opaque, homogeneous, and finely granular (Figs . 11 and 12) . Sections
from tissue sections incubated first for acid phosphatase activity and then for DAB oxidation at pH
9 clearly demonstrate that all large cytoplasmic
bodies, of all proximal convoluted tubule cells,
which are not apical vacuoles or mitochondria are
either lysosomes or peroxisomes (Figs . 11 and 12) .
The lysosomes in P I and P 2 cells are large and
show a somewhat more apical distribution than do
the peroxisomes (Fig . 11) . Lysosomes of P 3 cells
are much smaller than the peroxisomes, fewer in
number and more frequently seen near the
Golgi apparatus (Fig . 12) . Acid phosphatase ac-
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FIGURE 6
Published August 1, 1969
tivity is observed in P 3 cells in GERL (confirming
the circular profiles, suggesting that the circular
Elizabeth McDowell, personal communication),
profiles represent cross-sections of the tubules . The
and in the Golgi saccule facing GERL .
Peroxisomes, visualized through DAB oxidation,
tubular elements are often seen to be continuous
with the blebs and tails (Figs . 15 and 16) . Follow-
appear as spherical or frequently irregularly
ing DAB incubation, peroxisomes show a less elec-
shaped structures (Figs . 9-14) . The irregularity in
tron-opaque, slightly eccentric "core" (Figs . 9-16) .
shape may take the form of slight blebs or tail-like
In cytochemically stained light microscopic
extensions (Figs. 15 and 16) . The latter probably
preparations, what appear to be large peroxisomes,
correspond to the "protrusions" previously re-
2
ported (11, 62) . Any one peroxisome image may
cells, particularly in the P 3 segment . In light of the
have but a single bleb, or many blebs and tails
electron microscopic images, in which no peroxisome exceeds 1 .25 ju in diameter, these would ap-
giving the appearance of Chinese figures (Fig . 15) .
Rodlike DAB-positive profiles (Fig . 15) are inter-
µ
in diameter, are observed at the base of the
pear to be clusters of the organelles grouped so
preted to be sections of tails in which the main
closely together (Figs . 1 I and 13) as to be indistin-
bodies of the peroxisomes lie outside the plane of
guishable in the light microscope
section .
The cytochemical DAB procedure also enhances
DISCUSSION
visualization of internal structure in the peroxi-
The Graham and Karnovsky peroxidase procedure
somes . Circular profiles or tubules composed of
(19) was modified by Novikoff and Goldfischer
parallel dense lines are noted at the periphery of
many peroxisomes (Fig . 16) . The distance between
kidney, liver, and functional hepatomas for both
edges of the tubules corresponds to the diameter of
light and electron microscopy . Optimal visualiza-
(44) to visualize peroxisomes in aldehyde-fixed
M . E. BEARD AND A. B . NOVIKOFIr
Peroxisome Distribution
507
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FIGURE 8 10-µ frozen section of calcium formaldehyde-fixed kidney incubated in DAB medium ptl 9
for 30 min at 37 ° C . Peroxisomes (short arrow) of P3 cells are round, darkly stained granules more abunddant in the basal portion of the tubule cells than the apical portion . The elongate mitochondria are
lightly stained in cells of the thick limbs of Henle's loop and of the PI and P2 segments (long arrows) .
X 430 .
Published August 1, 1969
observation that all peroxisomes oxidize DAB
(electron microscopy) virtually eliminate the possibility that renal peroxisomes are heterogeneous
with regard to these two oxidative activities . By
incubating sections for both acid phosphatase and
DAB oxidation, it is shown that all large cytoplasmic bodies which are not mitochondria are
either peroxisomes or lysosomes . This is true as
well of rat liver hepatocytes (44) .
It has been suggested (16, 21, 46, 63) that
peroxisomes of hepatocytes form from expanded
regions of endoplasmic reticulum . Ericsson and
coworkers (11, 62) suggested the possible continuity of the peroxisome projections with the
endoplasmic reticulum (ER) in kidney . The absence of DAB reaction product in endoplasmic reticulum of the neonatal (Beard, unpublished)
as well as the adult kidney may indicate that, if
continuities of ER with renal peroxisomes exist, the
level of catalase or other DAB oxidant in the ER is
too low to be detected by the DAB pH 9 procedure .
The peroxisome core seen by us in incubated
material does not appear to be darker than the
dense core present in unincubated osmium tetroxide-fixed material (11, 35, 63) . It is possible that
the cores demonstrated in osmium tetroxide-fixed
tissue and in glutaraldehyde-fixed, DAB-incubated
tissue correspond, and that the material or structure in this central region does not oxidize DAB . It
is possible that the lack of DAB reaction product
in the center of microbodies reflects limited penetration of DAB into the organelle ; however, this
seems unlikely . The significance of the linear or
clustered arrangement of many peroxisomes and
Portion of a PI or P2 cell is shown . 25-ß nonfrozen section of glutaraldehydefixed kidney incubated in DAB medium pH 6 for 60 min at 37 äC . The thin section was
not stained . The mitochondria (Mt) are long and oriented perpendicularly to the base of
the cells . Reaction product is present in the cristae (arrows) . A light deposit of reaction
product is present in the peroxisomes (P) . Other labeled structures (unstained) are : basement membrane (BM), lysosome (L), brush border (BB) . A glutaraldehyde-fixation artifact is readily recognizable at A . Inset: Shows DAB reaction product in outer and inner
mitochondrial membranes (arrow) including the cristae . X 73,000 .
FIGURE 9
A portion of a P3 cell is shown . Tissue treated as in Fig. 9 . The thin section was
not stained . The mitochondria (Mt), most of which are elliptical or circular in outline rather
than long, show less reaction product than mitochondria of PI and P2 cells (see Fig . 9) .
Most cristae (long arrow) are lightly stained or unstained . Peroxisomes (P) are moderately
stained. Other labeled structures (unstained) are basement membrane (BM), brush
border (BB), endoplasmic reticulum (ER, short arrow), and nucleus (N) . X 15,000 .
FIGURE 10
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THE JOURNAL OF CELL BIOLOGY . VOLUME 42, 1969
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tion involved increasing the temperature to 37 ä C,
performing the reaction at alkaline pH (8-9), and
using greater concentrations of DAB and H 202 .
Independently, Hirai (22) used the GrahamKarnovsky procedure to visualize peroxisomes in
liver. The inhibition of the peroxisome reaction by
3-amino-1 ,2,4,triazole reported by Hirai (22)
and Novikoff and Goldfischer (45), and subsequently by Fahimi (17), as well as direct observations on catalase by Hirai (22), show catalase to be
involved in DAB oxidation by peroxisomes . In
contrast, mitochondrial oxidation of DAB is inhibited by cyanide (22, 45) . The reaction probably
depends upon the cytochromes in these organelles .
In the present study, the light microscopic
tetrazolium method was applied successfully, with
both formaldehyde- and glutaraldehyde-fixed tissue (see references 1, 2 where only the former
fixative was used) . Peroxisomes were visualized in
all the cells of the proximal convoluted tubule and
were concentrated in the basal half of the cells .
These results are in agreement with the report of
Maunsbach (35) who used fluorescence and phasecontrast microscopy in his studies, and with that
of Ericsson and Trump (15) who studied toluidine
blue-stained, Epon-embedded sections, but do not
agree with the earlier reports (1, 2) in which
peroxisomes were described only in P 3 cells. The
failure to see them in P I and P 2 cells previously may
reflect better preservation of the organelles by
glutaraldehyde, greater sensitivity of the cytochemical method, and longer incubation times .
The identical staining pattern of peroxisomes
shown by a-hydroxy acid oxidase and DAB
oxidation procedures (light microscopy) and the
Published August 1, 1969
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509
Published August 1, 1969
their concentration in the base of the cells is
unclear.
This study focuses attention upon the cells in the
P 3 segment of the proximal convolution . These
cells have an abundance of large peroxisomes with
high levels of catalase, a-hydroxy acid oxidase,
and presumably of n-amino acid oxidase . In
contrast, their mitochondria are smaller and
unoriented when compared with those of P, and
P 2 cells, and apparently have considerably lower
levels of cytochromes, and other metalloproteins
which might oxidize DAB (see reference 51) . The
mitochondria of P3 cells stain lightly with the DAB
pH 6 medium ; they give a marked response to
the addition of cytochrome c to the medium (as do
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THE JOURNAL OF CELL BIOLOGY • VOLUME
the mitochondria in Henle's loop, and in P, and
P 2 cells) ; and they stain more lightly than those of
P, and P2 cells when sections are incubated for
either succinate tetrazolium reductase or NADH 2tetrazolium reductase activities (Table I ; see
references 52 and 54) . These observations suggest
that P3 cells have low levels of succinic dehydrogenase and NADH 2-cytochrome c reductase activities and possibly of cytochromes . They suggest
further that P3 cells utilize a different mode of
oxidative metabolism than do cells elsewhere in
the nephron . Peroxisomes are implicated in an
extramitochondrial regeneration of cytoplasmic
NADH 2 in Tetrah)'mena pyriformis (37) . Peroxisome
enzyme activity in this organism is increased
42, 1969
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Portions of two PI or P2 cells are shown . 25-µ nonfrozen section of glutaraldehyde-fixed
kidney incubated in CMP medium for 12 min at 37 °C, followed by incubation in DAB medium pH 9
for 60 min at 37°C . Thin section stained with lead . Coarse, irregular deposits of lead phosphate are concentrated in large lysosomes (L), most of which are located in the basal portion of the cells. Small lead
phosphate deposits surrounding the lysosomes probably have resulted from diffusion of the reaction
product (or possibly enzyme) from the organelles. The more homogeneous DAB reaction product is
localized in peroxisomes (P) . Generally, an eccentric region in the peroxisomes (long arrow) shows little,
if any, reaction product . The peroxisomes are usually smaller than the largest lysosomes in the P1 and
P2 cells . A few mitochondria (Mt) show reaction product in cristae (short arrow) . Basement membrane
(BM) . X 11,500 .
FIGURE 11
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Portions of two P3 cells are shown . Tissue treated as in Fig. 11 . Thin section stained with
lead. The coarse lead phosphate deposits mark lysosomes (L) . The lysosomes are smaller and more apical
than in PI and P2 cells (see Fig . 11) . Lead phosphate is also seen in Golgi saccules (G) and has been observed in GERL (not illustrated) . The small lead phosphate deposits in the brush border (BB) may be
diffusion artefact, or may reflect the presence of other phosphatases . The DAB reaction product marks
the peroxisomes (P) . Note the lighter, slightly eccentric inner areas (arrow) . Peroxisomes appear circular
or slightly irregular, and most of them are situated more basally than most lysosomes . A linear arrangement of peroxisomes is commonly seen in some regions ; this is more evident in P1 or P2 cells (see Fig .
13) . No reaction product is found in the Initochondria (Mt), nuclei (N), and basement membrane (BM) .
X 6,800 .
FIGURE 12
when normal oxidation is suppressed and glyconeogenesis is induced (27) . In mammals, peroxisomes have been identified only in tissues in which
glyconeogenesis occurs, i .e . liver and kidney (9,
10) . Organelles of the size of mammalian peroxisomes and containing a similar complement of
enzymes have been identified in plants (61) .
Tolbert et al . (61) suggest that peroxisomes are
involved in an oxidative process (photo-respiration) different from mitochondrial oxidation .
Other differences between P 3 cells and P, and
P2 cells are the less numerous and much less deep
basal infoldings of the plasma membrane in P 3
cells ; the somewhat higher alkaline phosphatase
M. E.
activity (26, 28, 64) and lower nucleosidephosphatase activity (41) of the brush border ; smaller
and more apical lysosomes ; and stronger staining
for endoplasmc reticulum-associated nucleoside
diphosphatase (see Novikoff et al . [43] for an
association of this enzyme activity and cells possessing glucuronyl transferase activity implicated
in transport and secretion of conjugated glucuronides) . Previous studies (7, 8, 13, 19, 20, 30,
31, 35, 38-40, 53-60) indicate that resportion of
glucose and large protein molecules occurs to a
much lesser extent in the P 3 segment than in PI
and P2 segments . The manner in which cytological
differences between the P 3 and P1 , P2 cells are cor-
BEARD AND A.
B.
NOVIKOFF
Peroxisome Distribution
511
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FIGURE 13 Portion of a P1 or P2 cell is seen . 25-µ nonfrozen section of glutaraldehyde-fixed kidney incubated in DAB medium pH 9 for 60 min at 37°C . Thin section lead-stained . DAB reaction product
marks peroxisomes (P) . Most peroxisomes appear irregularly circular in outline and show lighter eccentric inner areas . Pr indicates what are probably cross-sections of peroxisome projections . Reaction product is also seen in the cristae (long arrow) of mitochondria . Any slight reaction product that might
be present in mitochondrial membranes is masked by lead staining . Irregular foci inside lysosomes
(L) show reaction product (short arrow) . No reaction product is present in endoplasmic reticulum
(ER), Golgi saccules (G), and plasma membrane (PM) . X 28,000 .
5 12
Published August 1, 1969
Foto . 15 Portion of a Pl or P2 cell is seen . Tissue treated as in Fig . 13 . Thin section stained with lead .
DAB reaction product is concentrated at the periphery of peroxisomes (P) . Irregular profiles and tubular
extensions (Pr) of peroxisomes are shown . Some extensions appear isolated (short arrow) from peroxisomes, probably because the central portion of the organelle lies outside the plane of the thin section .
Reaction product is seen in cristae (long arrows) of mitochondria (Mt) (see Fig. 13) . The endoplasmic
reticulum (ER) and nucleus (N) show no reaction product . X 28,000 .
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Portion of a P3 cell is seen . Tissue treated as in Fig . 13 . Thin section stained with lead .
Heavy reaction product is present at the periphery of the numerous peroxisomes (P) and only occasionally in the cristae (arrow) of some mitochondria (MI) (compare with Fig . 13 for differences in mitochondrial length) . Peroxisomes of this segment often occur in clusters (see Fig . 12) . No reaction product is
present in basement membrane (BM), endoplasmic reticulum (ER), and nucleus (N) . X 20,000 .
FIGLTE 14
Published August 1, 1969
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514
THE JOURNAL OF CELL BIOLOGY
•
VOLUME 42, 1969
Published August 1, 1969
related
physiological
We are grateful to Mrs . Regina Dominitz for
activities of the segments remains to be established .
with
differences
in their
assistance with the cytochemical preparations ; to
Mr . Nelson Quintana for assistance in electron mi-
This work was supported by United States Public
Health Service Post-doctoral Fellowship 1F2-GM36065-01 to Dr . Beard, and USPHS Research Grant
5-RO1-CA-06576-06 to Dr . Novikoff. Dr . Novikoff is
a recipient of the USPHS Research Career Award
5-K6-CA-14,923 from the National Cancer Institute .
croscopy ; and to Mr . Jack Godrich for preparation
of the photographs .
Received for publication 13 February 1969, and in revised
form 2 April 1969 .
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THE JOURNAL OF CELL BIOLOGY . VOLUME 42, 1969