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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 501 Downloaded from on June 15, 2017 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. 502 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 Downloaded from on June 15, 2017 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 503 Downloaded from on June 15, 2017 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 Published August 1, 1969 Downloaded from on June 15, 2017 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 5 05 Downloaded from on June 15, 2017 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 5 06 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- 1969 Downloaded from on June 15, 2017 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 Downloaded from on June 15, 2017 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 10 508 THE JOURNAL OF CELL BIOLOGY . VOLUME 42, 1969 Downloaded from on June 15, 2017 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 Downloaded from on June 15, 2017 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 510 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 Downloaded from on June 15, 2017 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 Published August 1, 1969 Downloaded from on June 15, 2017 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 Published August 1, 1969 Downloaded from on June 15, 2017 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 . Downloaded from on June 15, 2017 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 Downloaded from on June 15, 2017 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 . REFERENCES 1 . ALLEN, J . M., and M . E . BEARD . 1965 . a-Hydroxy acid oxidase : Localization in renal microbodies. Science . 149 :1506 . 2 . ALLEN, J . M ., M . E . BEARD, and S . KLEINBERGS . 1965 . The localization of a-hydroxy acid oxidase in renal microbodies . J. Exp . Zool . 160 :329. 3 . BAKER, J . R. 1946 . The histochemical recognition of lipine . Quart . J . Micr. Sei. 87 :441 . 4. BARKA, T ., and P . J . ANDERSON . 1963 . Histochemistry : Theory, Practice and Bibliography . 6. 7. 8. fragments of single rabbit nephrons . Amer. J. Physiol . 210 :1293 . 9 . DE DuvE, C . 1963 . The separation and characterization of subcellular particles . In The FIGURE 16 Portion of a P3 cell is DAB medium pH 8.6 for 60 min tion product in peroxisomes (P) reactivity of the peroxisomes at 11 . 12 . 13 . microscopy and cytochemistry of acid phosphatase . II . Electron microscopy . Lab . Invest . 14 :1 . 14 . ERICSSON, J . L . E ., B. F . TRUMP, and J . WEIBEL . 1965 . Electron microscopic studies of the proximal tubule of the rat kidney . II . Cytosegresomes and cytosomes . Their relationship to each other and to the lysosome concept . Lab . Invest . 14 :1341 . 15 . ERICSSON, J . L. E ., and B . F . TRUMP . 1966 . Electron microscopic studies of the epithelium of the proximal tubule of the rat kidney . III . Microbodies, multivesicular bodies and the Golgi apparatus . Lab . Invest . 15 :1610 . seen . 25-µ nonfrozen section of glutaraldehyde-fixed kidney incubated in at 27 ° C . Thin section lead-stained . Examined at 50 kv . The DAB reacappears granular, possibly due to overstaining with lead or to lower pH 8 .6 than at pH 9, seen in all previous figures . The projections con- tinue into the body of the organelles where they appear in circular (C) or tubular (T) profiles . Inset: A portion of another P3 cell . Thin section lead-stained . Some peroxisomes are sectioned sufficiently perpendicularly for the limiting membrane to be seen (arrow) . ER indicates endoplasmic reticulum that often surrounds the peroxisomes (P) . Mitochondrion (Mt) . One peroxisome shows an unusually light deposit of reaction product. X 48,000. M . E. BEARD AND A . B . NOVIKOFF Peroxisome Distribution 515 Downloaded from on June 15, 2017 5. Harper and Row Publishers, Inc ., New York. BAUDHUIN, P ., H . BEAUFAY, and C . DE DuvE . 1965 . Combined biochemical and morphological study of particulate fractions from rat liver . Analysis of preparations enriched in lysosomes or in particles containing urate oxidase, D-amino acid oxidase, and catalase . J. Cell Biol . 26 :219. BEARD, M. E ., and J . M . ALLEN . 1968 . A study of properties of renal microbodies of the rat . J . Exp. Zool . 168 :477 . BENNETT, C . M., B . M . BRENNER, and R. W . BERLINER . 1968 . Micropuncture study of the nephron function in the rhesus monkey . J . Clin . Invest . 27 :402 . BURG, M ., J . GRANTHAM, M . A . ABRAMOW, and J . ORLOFF . 1966 . Preparation and study of 10. Harvey Lectures . Academic Press Inc ., New York . DE DUVE, C ., and P. BAUDHUIN . 1966 . Peroxisomes (microbodies and related particles) . Physiol. Rev . 46 :323 . ERICSSON, J . L . E . 1964 . Absorption and decomposition of homologous hemoglobin in renal proximal tubular cells . An experimental light and electron microscopic study . Thesis. Acta Pathol . Microbiol . Scand. Suppl . 168 . ERICSSON, J . L . E ., and B. F . TRUMP . 1964 . Electron microscopic studies of the epithelium of the proximal tubule of the rat kidney . I . The intracellular localization of acid phosphatase. Lab . Invest . 13 :1427 . ERICSSON, J . L . E . 1965 . Transport and digestion of hemoglobin in the proximal tubule . I . Light Published August 1, 1969 16 . ESSNER, E . 1967 . Endoplasmic reticulum and the origin of microbodies . Lab . Invest . 17 :71 . 17 . FAHIMI, H . D . 1968 . Cytochemical localization of peroxidase activity in rat hepatic microbodies (peroxisomes) . J. Histochem . Cytochem . 16 :547 . phase in cells of microperfused single proximal tubules . J. Ultrastruct . Res . 16 :1 . 31 . MAUNSBACH, A. B. 1966 . Absorption of 1125_ 18 . GOMORI, G . 1952 . Microscopic Histochemistry . University of Chicago Press, Chicago . 19 . GRAHAM, R . C ., JR ., and M. J . KARNOVSKY . 1966 . The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney . Ultrastructural cytochemistry by a new technique . J. Histochem. Cytochem . 14 :291 . 20 . GRIFFITH, L . D ., R. E. BULGER, and B . F . TRUMP . microscopic autoradiography and histochemistry . J. Ultrastruct . Res . 15 :197 . 32 . MAUNSBACH, A . B . 1966 . Observations of the segmentation of the proximal tubule in the rat kidney . Comparison of results from phase contrast, fluorescence and electron microscopy . J. Ultrastruct . Res . 16 :239 . 33 . MAUNSBACH, A . B . 1966 . Observations on the ultrastructure and acid phosphatase activity of the cytoplasmic bodies in rat kidney proximal tubule cells : with a comment on their classification . J. Ultrastruct. Res . 16 :197 . 34 . MAUNSBACH, A . B. 1966 . The influence of different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubule cells . I . Comparison of different perfusion fixation labelled homologous albumin by rat kidney proximal tubule cells . A study of microperfused single proximal tubules by electron 35 . MAUNSBACH, A . B. 1966 . The ultrastructure of renal proximal tubule cells as related to protein absorption. Thesis, Department of Zoology, University of California, and Department of Anatomy and Histology, Karolinska Institutet, Stockholm, Sweden . 24. HRUBAN, Z ., and H. SWIFT. 1964 . Uricase : localization in hepatic microbodies. Science . 146 : 1316 . 25 . HRUBAN, Z ., H . SWIFT, and A . SLESERS . 1966 . Ultrastructural alterations of hepatic microbodies . Lab . Invest . 15 :1884 . 36 . MILLONIG, G . 1962 . Further observations on a phosphate buffer for osmium solutions in fixation . Proceedings of the Fifth International Congress for Electron Microscopy, Philadelphia, 1962. Sydney S. Breese, Jr ., editor . Academic Press Inc ., New York . 2 :8. methods and of glutaraldehyde, formaldehyde and osmium tetroxide . J. Ultrastruct . Res. 15 : 242 . 26 . JACOBSEN, N . O ., F . JORGENSEN, and A. O. THOMSEN . 1967 . On the localization of some phosphatases in three different segments of the proximal tubules in the rat kidney . J. Histochem . Cytochem . 15 :456 . 37 . MÜLLER, M., J . F. HOGG, and C . DE DUVE . 1968 . Distribution of tricarboxylic acid cycle enzymes between mitochondria and peroxisomes in Tetrahymena pyriformis . J . Biol . Chem . 243 : 5358 . 27 . LEVY, M., and A . HUNT . 1968 . L-a-hydroxy acid 38 . NACHLAS, M . M ., K .' C . Tsou, E . DESOUZA, oxidase activity in Tetrahymena . Change C-S . CHENG, and A . M. SELIGMAN . 1957 . with physiological state . J. Cell Biol. 34 :911 . Cytochemical demonstration of succinic de28 . LONGLEY, J . B ., and E . R . FISCHER . 1954 . Alkaline phosphatase and the periodic acid Schiff reaction in the proximal tubule of the vertebrate kidney . Anat . Rec . 120 :1 . 29 . LUFT, J . H . 1961 . Improvements in epoxy resin embedding methods . J. Biophys . Biochem . Cytol . 9 :409 . 30 . MAUNSBACH, A . B. 1966 . Absorption of ferritin by rat kidney proximal tubule cells . Electron microscopic observations of the initial uptake 516 hydrogenase by the use of a new p-nitrophenyl substituted ditetrazole . J. Histochem . Cytochem . 5 :420 . 39 . NEUSTEIN, H . B . 1967 . Hemoglobin absorption in the proximal tubules of the kidney in the rabbit. J. Ultrastruct. Res. 17 :565 . 40 . NEUSTEIN, H . B . 1966. Hemoglobin absorption by proximal tubule cells of the rabbit kidney . A study of electron microscopic autoradiography . J. Ultrastruct. Res. 16 :141 . THE JOURNAL OF CELL BIOLOGY . VOLUME 42, 1969 Downloaded from on June 15, 2017 1967 . The ultrastructure of the functioning kidney. Lab . Invest. 16 :220. 21 . HAGIWARA, A., T . SUZUKI, and F . TAKAKI . 1961 . Electron microscopic cyto-histopathology . VIII . Electron microscopic studies of the liver . Studies on the origin of the so-called "microbodies" as a precursor of rat liver mitochondria and their relation to the secretion granules of the bile . Jikeikai Med. J. 8 :51 . 22 . HIRAI, K . 1968 . Specific affinity of oxidized amine dye (radical intermediator) for heme enzymes . Study in microscopy and spectrophotometry . Acta Histochem . Cytochem . 1 :43 . 23 . HRUBAN, Z ., and M . REIGHCIGL . 1967 . Comparative ultrastructure of microbodies . Fed. Proc . 26 :1428 . Published August 1, 1969 41 . NoviKOFF, A . B . 1963. A cytochemical study of the "third cell" in the mammalian glomerulus . J. Cell Biol . 19 :88A . (Abstr .) 42 . NovIKOFF, A . B . 1963 . Lysosomes in the physiology and pathology of cells : contribution of staining methods . In Ciba Foundation Symposium on Lysosomes. A . V. S . DeReuck and M. P . Cameron, editors . Little, Brown and Co., Boston . 36 . 43 . NOVIKOFF, A . B ., E . ESSNER, S . GOLDFISCHER, and M. HEUS . 1962 . Nucleoside-phosphatase activities of cytomembranes . In Symposia of the International Society for Cell Biology, Academic Press Inc ., New York. 1 :149 . 44 . NOVIKOFF, A . B ., and S . GOLDFISCHER . 1968 . Visualization of microbodies for light and electron microscopy . J. Histochem . Cytochem . 16 :507 . 45 . NOVIKOFF, A . B ., and S . GOLDFISCHER . 1968 . Visualization of microbodies for light and 1963. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol . 17 :19 . 51 . SELIGMAN, A . M ., M. J . KARNOVSKY, H . L . WASSERKRUG, and J . S . HANKER . 1968. Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB) . J. Cell Biol . 38 :1 . 52 . SELIGMAN, A. M ., H . UENO, Y. MORIZONO, H. L . WASSERKRUG, L. KATZOFF, and J . S. HANKER . 1967. Electron microscopic demonstration of dehydrogenase activity with a new osmiophilic ditetrazolium salt (TC-NBT) . J. Histochem. Cytochem. 15 :1 . 54 . STERNBERG, W . H ., E . FARBER, and C . E . DUNLAP . 1956 . Histochemical localization of specific oxidative enzymes . II . Localization of diphosphopyridine nucleotide diaphorases and the succindehydrogenase system in the kidney . J. Histochem . and Cytochem . 4 :266. 55 . STRAUS, W . 1962. Cytochemical investigation of phagosomes and related structures in cryostat sections of kidney and liver of rats after intravenous administration of horseradish peroxidase . Exp . Cell Res. 27 :80. 56 . STRAUS, W . 1963 . Comparative observations on lysosomes and phagosomes in kidney and liver of rat after administration of horseradish peroxidase . In Ciba Foundation Symposium on Lysosomes . Little, Brown and Co., Boston . 151 . 57 . STRAUS, W. 1964 . Cytochemical observations on the relationship between lysosomes and phagosomes in kidney and liver by combined staining for acid phosphatase and intravenously injected horseradish peroxidase. J. Cell Biol . 20 : 497 . 58 . STRAUS, W . 1964. Occurrence of phagosomes and phago-lysosomes in different segments of the nephron in relation to the reabsorption, transport, digestion and extrusion of intravenously injected peroxidase. J. Cell Biol. 21 :295 . 59. STRAUS, W . 1967 . Methods for study of small phagosomes and of the relationship to lysosomes with horseradish peroxidase as a "marker protein ." J. Histochem. Cytochem . 15 :375 . 60. STRAUS, W . 1967 . Changes in intracellular location of small phagosomes (micropinocytotic vesicles) in kidney and liver cells in relation to time after injection and dose of horseradish peroxidase . J. Histochem . Cytochem . 15 :381 . 61 . TOLBERT, N . E ., A. OSER, T . KISAKI, R . H. HAGEMAN, and R . K . YAMAZAKI . 1968 . Peroxisomes from spinach leaves containing enzymes related to glycolate metabolism . J. Biol . Chem . 243 :5179 . 62 . TRUMP, B. F ., and J . L . E . ERICSSON . 1965 . The effect of the fixative solution on the ultrastructure of cells and tissues . A comparative analysis with particular attention to the proximal convoluted tubule of the rat kidney. Lab. Invest. 14 :507 . 63. TSUKADA, H ., Y. MOCHIZUKE, and S . FUJIWARA. 1966 . The nucleoids of rat liver cell microbodies . Fine structure and enzymes . J. Cell Biol . 27 :449. M. E . BEARD AND A . B . NOVIKOFF Peroxisome Distribution 517 Downloaded from on June 15, 2017 electron microscopy. Presentation at the 19th Annual Meeting of the Histochemical Society, April 1968. 46 . NovIKOFF, A . B., and W-Y. SHIN . 1964 . The endoplasmic reticulum in the Golgi zone and its relation to microbodies, Golgi apparatus and autophagic vacuoles in rat liver cells . J. Microsc . 3 :187. 47 . NOVIKOFF, A . B ., W-Y. SHIN, and J. DRUCKER . 1961 . Mitochondrial localization of oxidative enzymes : staining results with two tetrazolium salts . J. Biophys. Biochem . Cytol. 9 :47. 48. REYNOLDS, E. S . 1963 . The use of lead citrate at high pH as an electron-opaque stain in electron microscopy . J. Cell Biol . 17 :208 . 49. RHODIN, J . 1954. Correlation of ultrastructural organization and function in normal and experimentally changed proximal convoluted tubule cells of mouse kidney . Thesis. Aktiebolaget Godvil, Stockholm, Sweden . 50. SABATINI, D . D ., K . BENSCH, and R . J . BARRNETT. 53 . SHNITKA, T . K . 1966 . Comparative ultrastructurc of hepatic microbodies in some mammals and birds in relation to species differences in uricase activity. J. Ultrastruct. Res . 16 :598. Published August 1, 1969 64 . WACHSTEIN, M . 1955 . Histochemical staining reactions of the normally functioning and abnormal kidney . J. Histochem. Cytochem . 3 :246 . 65 . WACHSTEIN, M., and E . MEISEL . 1956 . On the histochemical demonstration of glucose-6phosphatase. J. Histochem . Cytochem . 4 :592 . 66 . WACHSTEIN, M., and E . MEISEL . 1957 . Histochemistry of hepatic phosphatases at a physio- logical pH with special reference to the demonstration of bile canaliculi . Amer . J. Clin . Pathol . 27 :13 . 67 . WALKER, D ., and A . SELIGMAN, 1963 . The use of formalin fixation in the cytochemical demonstration of succinic and DPN- TPN-dependent dehydrogenases in mitochondria . J. Cell Biol . 16 :455 . Downloaded from on June 15, 2017 5 18 THE JOURNAL OF CELL BIOLOGY . VOLUME 42, 1969