Localization of intravenously injected horseradish peroxidase in the cells of the convoluted tubules of rat kidney

600

LOCALIZATION

OF INTRAVENOUSLY

PEROXIDASE

IN THE TUBULES

CELLS

INJECTED OF THE

OF RAT

HORSERADISH

CONVOLUTED

KIDNEY

W. STRAUS’ Laboratory

qf Physiological of Animal

Chemistry, University qf Louvain, and Laboratory Morphology, University of Brussels, Belgium Received May 8, 1960

THEfate

of intravenously injected horseradish peroxidase can easily be determined in animal cells, by calorimetry of tissue extracts and of isolated fractions in vitro [IO, 111, and cytochemically in situ [la]. By these methods, granules (“phagosomes”) which are characterized by the ability to concentrate foreign proteins have been identified in the cells of various tissues [ll, 121. Granules of this type which vary in diameter from the limit of microscopic visibility up to 5 p, have been isolated from the kidney cells of rats and have been found to contain high concentrations of hydrolytic enzymes [7, 81. These same enzymes have been discovered by de Duve et al. [2] in the “lysosomes” of liver. In an extension of the previous work, the changes taking place in the kidney cells of rats during the reabsorption and excretion of horseradish peroxidase are now being analyzed in greater detail by a correlation of cytochemical and biochemical methods. In the meantime, Novikoff [3] and Wachstein et al. [14] have also applied our method to the kidney. This has prompted the present brief report on our cytochemical observations. A more detailed account of these and of the corresponding biochemical data will be given later. Twenty to fifty milligrams of a commercial horseradish peroxidase preparation (Sigma Chemical Company, Saint Louis, Missouri, U.S.A.) were injected into the femoral veins of male albino rats weighing 200 to 250 g. After periods varying from a few minutes to a few hours, the kidneys were perfused in situ with cold sucrose solution, and small pieces of the cortex were squashed on a microscope slide with a coverslip and incubated for l-3 minutes in cold benzidine solution in 70 per cent ethyl alcohol [la]. In some experiments, the kidneys (cortices) were homogenized incompletely in 30 per cent sucrose solution, the non-homogenized residue was separated by low speed centrifugation, and a little of the sediment was treated as above. Details of the procedures and the advantages and disadvantages of using squashed fresh tissue will be mentioned in a later report. The following observations were made. Within a few minutes after administration of peroxidase, the regions of the brush border and of the basement membrane are Peroxidase penetrates a short distance into the strongly stained with benzidine. apical cytoplasm where it forms blue-staining strands along the lumen and in the perinuclear regions (Figs. l-3). This localization suggests reabsorption of the foreign 1 This investigation was supported by an Established Association and by a research grant from the National Health Service (No. C-2485). Experimental

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Investigatorship of the American Heart Cancer Institute, United States Public

Localization

of injected peroxidase in kidney

601

protein from the lumen. A second characteristic site of concentration of peroxidase is found on benzidine-positive membranes or fibrils extending from the basal to the apical regions of the cells (Figs. 4-5). The membranes or fibrils run roughly parallel, separated by an estimated distance of 0.2 to 0.5 p. Peroxidase often appears concentrated on these fibrils in very small granules (Fig. 4). It may be suggested tentatively that these fibrils or membranes represent the membranes of the endoplasmic reticulum (4, 5) and the infoldings of the apical and basal cell membranes as observed by electronmicroscopy [l, 61. After injection of peroxidase, these membranes might join and thus form a canalicular system connecting the base of the cells with the lumen. The nuclear membranes which are also stained by benzidine (Figs. 1,7), seem to be a part of this system. Although all these lamellar structures are submicroscopic, they seem to become coarser and visible in the microscope because of the blue pigment formed by the reaction of peroxidase with benzidine. In addition to the strands and membranes mentioned, benzidine stains discrete canaliculi perhaps representing intercellular spaces. It is often difficult to distinguish these canaliculi from the bluestaining strands mentioned above which may extend in the cytoplasm adjacent to the canaliculi all along their course. The canaliculi can be recognized when they show two blue lines (membranes) bordering a narrow white line (lumen) after staining with benzidine (Fig. 6 this report and Fig. 3 1131). Large “phagosomes” (l-5 p diameter) were observed to develop between the rows of the fine parallel membranes and between the membranes of the discrete canaliculi (Fig. 6), and in the strands of cytoplasm adjacent to these membranes and canaliculi and adjacent to the apical cell membranes (Figs. l-3). Between 10 and 60 minutes after administration of peroxidase, a condensation of finely granular material into large inclusion bodies takes place along these strands and membranes in the apical cytoplasm (Figs. l-3). This development explains the arrangement of the “droplets” in rows, one behind the other, near the cell membranes as reported previously [12]. The bodies can often be seen to protrude from the surface membranes into the lumen as if it were a process of secretion (Fig. 3). A part of the large “phagosomes” probably are extruded into the lumen. Another part remains in the tissue for several days. \$Thereas the large phagosomes were observed to develop exclusively in the apical regions of the cells during the first hour after administration of peroxidase (Figs. l&3), they were also found in a more basal location at later periods (Fig. 7). As mentioned, peroxidase appears to be concentrated in rows of very small granules on the fine, parallel membranes which connect the base of the cells with the lumen (Fig. 4). In accordance with the previous hypothesis, it is suggested that these small phagosomes serve in the transport of peroxidase [12], and that they take part in the formation of the large inclusion bodies [9]. Since the “phagosomes” contain both the exogenous protein (horseradish peroxidase) [lo] and endogenous proteins (hydrolytic enzymes) [X, 91, their development may be considered to be a combination of the processes of pinocytosis and secretion. The possible significance of the granules, membranes, canaliculi and strands, for transport, secretion and pinocytosis will be discussed in some detail later. I am greatly indebted to Professor Professor Christian de Duve, University have accorded me in their laboratories.

Jean Brachet, University of Brussels, and of Louvain, for the hospitality which they

Bxperimentul

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Experimental

Cell Research 20

Localization

of injected peroxidase in kidney

603

REFERENCES I. (:LARK, .Ju., S. I.., .I. Uiophys. ~iochem. Cytol. 3, 349 (1957). 2. DE kVE, c., PRESSMAS, B. (:., GIANKTTO, R., WATTIAUX, R. and APPELMASS, F., J. 60, 604 (1955). 3. NOWKOPF, A. B., in Riology of Pyclonephritis. f-:. I,. Quimi, E. H. Kass, Ms., Little. Co.. I3oston, 1960. 2. P.ZLAUE, G. R., ./. Ijiophys. Biochem. Cgfol. 2, Ko. 4, suppl., 85 (1956). 5. PoR~rEn. Ii. R., IInruey Lectures, 51, 175 (1957). 0. RHODIS. .J., Internafl. Reu. C!yfof. 7, 485 (1958) 7. STRAUS, U’., J. Bid. Chem. 207, 745 (1954). X. ~~~~ .I. Jliophgs. Ziiochern. Cyfof. 2, 513 (1956). 9. - ~ ibid. 3, 933 (1957). IO. ~ -- ibid. 3, 1037 (1957). 11. -~~ ibid. 4, 541 (1958). 12. ~ibid. 5, 193 (1959). 13. --~ Exptf. Cell Hesetrrch. In press. XI., MEISEL, E. and FALCOS, C., J. Hisfochem. Cgfochem. 7, 128 (1959). 11. \V.~CHSTEIS,

Ijiochem. Rrown

$

Figs. 1 and 2.PIknzidinc-positive strands in apical cytoplasm and perinuclcar regions of (distal?) tubule cells; 30 minutes (Fig. 1) and 60 minutes (gig. 2) after intravenous administration of horseradish pcroxidasc. Note that the inclusion bodies, aligned in rows one behind the other (arrows). dcvclop on the strands. Magnification, x 1350. Fig. 3.-- Devclopmcnt of inclusion bodies in bcnzidinc-positive strands of cytoplasm in the apical regions of proximal convoluted tubule cells; 30 minutes after administration of peroxidasc. Note that some inclusion bodies protrude into the lumen (arrows) as in a process of secretion. and that the basement mrmbranc is stained. Magnification, x 950. I:ig. 4.~l~cnzidine-positive strands and fine, parallel fibrils or membranes; 30 minutes after administration of pcroxidase. Note that some of the strands contain developing inclusion bodies. and that the fine, parallel fibrils extend from the bascmcnt membrane and have a finely granular appearance. Magnification, y 950. Fig. 5.Plknzidine-positive membranes or fibrils extending from the basal to the apical regions of the cells; 4 hours after administration of peroxidase. The inclusion bodies, located between the fibrils, arc not in focus. The fine blue granularity is due to artifacts after too long incubation with benzidine. Alagnification, ‘i 950. Fig. fi.---Rcnzidine-positive minutes after administration

canaliculum, of peroxidase.

showing small Magnification,

phagosomes x 1350.

on its membranes;

60

i’ig. 7.PlSenzidine-positive, large phagosomes, some of which located near the bascmcnt mcnbrane (arrow); 3 hours after administration of peroxidase. Note in background fine, benzidinepositive membranes of granular appearance, including nuclear membranes. Magnification. j: 1350. Figs. I, 2. l-7.--Squash ments

(see

preparations

of fresh tissue; I’ig. 3: squash preparation

of tissue frag-

test).

Experimerlfrtl

Cell Xrsrctrch 20