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Histochemical localisation of adenosine triphosphatase activity in adult and newborn rat kidneys at the.. Lim, Wan Cheng 1969-12-31

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THE HISTOCHEMICAL LOCALISATION OF ADENOSINE-TRIPHOSPHATASE ACTIVITY IN ADULT AND NEWBORN  RAT KIDNEYS AT THE ELECTRON MICROSCOPICAL LEVEL " by WAN CHENG LIM B.A., Wellesley College, 1968 A TRESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Anatomy We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1969 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree tha permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Anatomy  The University of British Columbia Vancouver 8, Canada Date November, 1969 i. ABSTRACT The. histochemical localisation of ATPase enzymatic activity at the level of the electron microscope was carried out on adult and newborn kidney tissue pre-fixed in 55^ gluta-raldehyde buffered with 0.1 M sodium cacodylate. Both the lead method at pH 7.<S and the calcium method at pH 9.4 were used. The effects of the modifiers PHMB and L-cysteine were also studied. In the adult rat kidney, the observations of other investigators on kidney ATPase activity were substantiated. Reaction precipitate was localised at the brush border of the proximal tubules, the membranes of the basal and lateral interdigitations of the proximal and distal tubules, and the plasma membranes of the podocytic foot processes. PHMB exerted an inhibitory effect on distal tubular activity at both pH 7.2 and pH 9.4, while cysteine was inhibitory only at pH 9.4. Glomerular ATPase activity was inhibited by PHMB and L-cysteine at pH 9.4. In the newborn rat kidney, ATPase enzymatic act- ' ivity was observed in the tubular elements as well as in the glomeruli. In the undifferentiated tubules, reaction product was abundant on the lateral membranes between indiv idual cells. The luminal and basal plasma membranes, which • were simple in contour, showed little or no accumulation of precipitate. However, as the microvilli became long and slender in the early stages of the differentiation of the brush border, there was a concomitant increase in the intens-ity-oof the ATPase enzymatic reaction. Similarly, reaction product became associated with the developing basal inter digitations. In the immature glomerulus, reaction precipit ate was most often observed where two sets of membranes were in apposition. With differentiation, enzymatic activity was localised primarily on the podocytic foot processes. The localisation of ATPase activity at pH 9.4 was found to be influenced by the time of pre-fixation in glutaraldehyde 0 while ATPase activity at pH 7.2 was not affected. At pH 7.2, neither tubular nor glomerular ATPase enzymatic activity responded to PHMB or L-cysteine. For both adult and newborn kidneys, the correlat ion between structure and function was briefly considered. The adult kidney is an important and efficient homeostatic organ. In urine formation various substances are transport ed across the cell membranes of the glomeruli and tubules. Ultrastructurally, the glomeruli and tubules show modifi cations characteristic of cells engaged in active trans port processes. There is a large increase in plasma memb rane surface area, as exemplified by the intricate inter-digitations of the podocytic foot processes, the elaborate basal and lateral infoldings, and the brush border of the proximal tubules. Much ATPase activity was found associated-with these plasma membranes. The newborn kidney is not as efficient as the adult kidney in maintaining body homeo stasis. It is not only functionally but also morphologically immature. Most of the tubules and glomeruli are undifferen tiated and do not show specialisations of the plasma membranes as seen in the adult kidney. There is also a relatively smaller amount of ATPase present in the newborn kidney. For both adult and newborn kidneys, it was postulated that at least two types of ATPases with different pH optima are present on the plasma membranes of the tubules and glomeruli. iii. TABLE OF CONTENTS Page I Introduction 1 II Materials and methods 7 A. MaterialsB. Pre-fixation in glutaraldehyde 7 C. Incubation for histochemistry 9 (a) Preparation of incubating medium at pH 7.2 9 (b) Preparation of incubating medium at pH 9.4 9 D. Effect of modifiers 10 E. Controls 1P. Post-fixation in osmium 11 G. Eleo'tron microscopy 1III Observations 12 A. Morphological basis for histochemical interpretations 12 (a) Adult kidney(b) Newborn kidney 15 B<. Effect of fixation on ultra structure 17 (4) Adult kidney 1(b) Newborn kidney 8 C. Effect of fixation on enzymatic activity 19 D. Enzymatic activity of the adult kidney 21 (a) pH 7.2 - lead method 2(b) pH 9.4 - calcium method 23 E. Enzymatic activity of the newborn kidney 2(a) pH 7.2 - lead method 23 (b) pH 9.4 - calcium method 25 F. Effect of modifiers PHMB and L-cysteine on enzymatic activity 27 (a) Adult kidney(b) Newborn kidney 29 iv. $s Page G. Controls 30 H. Nuclear staining 3IV Discussion 2 A. General 3B!. Plasma membrane enzymatic activity 35 (a) Adult kidney 3(b) Newborn kidney 40 C. Summary of enzymatic activity of the adult and newborn kidney 42 D. Nuclear staining 44 E. Controls 45 V Illustrations 6 VI Bibliography 91 V. TABLES Page I Age of the animals and the length of fixation of the respective kidneys. 8 II The effect of fixation on enzymatic activity. 20 III The effect of the modifiers, PHMB and L-cysteine, on enzymatic activity of the adult kidney. 27 IV The effect of the modifiers, PHMB and L-cysteine, on enzymatic activity of the newborn kidney. 29 vi. ILLUSTRATIONS Figures Pages 1-4 Enzymatic activity of the adult kidney at pH 7.2. 47 - 50 5 - 10 Enzymatic activity of the newborn kidney at pH 7.2. 51 - 56 11 - 17 Enzymatic activity of the newborn kidney at pH 9.4. 57 - 63 18 - 25 Effect of modifiers on enzymatic activity of the adult kidney. 64-71 26 - 36 Effect of modifiers on enzymatic activity of the newborn kidney. 72-82 37 - 40 Controls with the Wachstein-Meisel method at pH 7.2 - adult kidney. 83 - 86 41 - 44 Controls with the Wachstein-Meisel method at pH 7.2 - newborn kidney. 87 - 90 vii. ACKNOWLEDGMENT I would like to express my appreciation to my advisor, Dr. William A. Webber, for his guidance through out the course of this investigation, and for suggestions and constructive criticisms in the preparation of the thesis; and to Mrs. Janice Blackbourn, for helpful hints in the techniques of electron microscopy. 1. I INTRODUCTION The localisation of enzymatic activity using "bio chemical methods on homogenised tissue fractions is a means of assaying quantitatively the various properties of enzymes under conditions approaching their natural state. These fract ionation studies, though useful and indispensable, have an inherent limitation. They show the association of enzymes with certain types of cellular components, but give no indic ation of the actual distribution of the enzymes within indi vidual cells or within groups of cells. At the present time, a histochemical or cytochemical technique for emzyme local isation represents the only way of determining the sites of enzymatic activity under in situ or near in situ conditions (Marches!, 1968). The main advantage of using a histochemical technique for enzyme localisation is that cellular relation ships are maintained in an intact tissue section. It is there fore possible to visualise the products of an enzymatic react ion in relation to specific cellular components, for example, mitochondria, nuclei or membranes. This has only been made feasible by vast improvements in the field of electron micro scopy. Histochemistry at the level of the electron microscope, opens up Large areas in cell ultrastructural research. We must however, bear in mind the limitations of existing* methods for the ultrastructural localisation of enzymatic activity. There needs to be adequate preservation of both the ultra-structure and the enzymatic activities of cells and tissues. Ideally, fresh tissue should be used, but this is not possible owing to extremely poor preservation of cell ultrastructure. A compromise is made by a brief pre-fixation in formaldehyde (Kaplan and Novikoff, 1959; Holt and Hicks, 1961), or more commonly, in glutaraldehyde (Sabatini et al., 1963; 1964), prior to incubation. The effect of fixation on the character istics of enzymes, whether quantitative or qualitative or 2. both., is not known at the present time. Besides fixation, the tissue undergoes rigorous conditions of dehydration, infiltr ation, embedding and polymerisation in an oven, in preparat ion for its examination under the electron beam (Sjostrand, 1967). How these processes affect the tissue are poorly, if at all, understood. Nevertheless, if these limitations are borne in mind, much information can be gleaned from electron microscopical histochemical data. Glenner (1968) defines the histochemical system as "that system in which intact tissue sections are incubated in a solution containing a substrate, and the enzyme-catalised formation of the final reaction product (a precipitate) can be demonstrated in situ by means of a variety of techniques including light, fluorescent, electron and interference micro scopy." Many histochemical methods, to date, are based on simple metal-salt precipitation reactions. The two common pro cedures for the histochemical localisation of adenosine tri phosphatase (ATPase) in tissue sections use lead ions (Wachs-tein and Meisel, 1957) or calcium ions (Padykula and Herman, 1955a, 1955b; Padykula and Gauthier, 1965) to form a precipi tate with the inorganic phosphate which is released by the enzymatic hydrolysis of the substrate in the incubation media, that is, adenosine triphosphate (ATP). The reaction product, which is electron dense and can be viewed directly under the electron microscope, is presumably deposited at, or very near to, the actual site of the enzymatic activity. This issue of whether the site of deposition of the reaction precipitate is the actual site of enzymatic activity, has not been solved yet, as many investigators stress (Otero-Vilardebo et al., 1963; Wachstein and Besen, 1964; Mao and Nakao, 1966). Most cells store energy in the form of high energy phosphate bonds in ATP (Stumpf, 1953). This energy can be re leased for use in performing various cellular activities by the enzyme ATPase. Therefore, the localisation of the enzyme ultrastructurally would perhaps lead to some understanding of the mechanisms underlying cellular function. Since the introduction of the lead (Wachstein and Meisel, 1957) and calcium (Padykula and Herman, 1955a, 1955b; Padykula and Gauthier, 1963) methods for the histochemical localisation of ATPase, many sites in various tissues have been demonstrated to possess this enzyme. This has been shown both with light microscopy (Padykula and Herman, 1955a, 1955h; Wachstein, 1955; Wachstein and Meisel, 1957; Wachstein et al., I960, 1962; Padykula and Gauthier, 1963; Tewari and Bourne, 1963a, 1963b; McClurkin, 1964; Wachstein and Besen, 1964; Far-quhar and Palade, 1966; Moses et al., 1966; Gauthier, 1967; Jacobsen et al., 1967; Brooke and Kaiser, 1969) and electron microscopy (Essner et al., 1958; Kaplan and Novikoff, 1959; Persijn et al., 1961; Lazarus and Barden, 1962; Ashworth et al., 1963; Oterb-Vilardebo et al., 1963; Torack'and Barrnett, 1963; Wachstein and Besen, 1963; Goldfischer et al., 1964; Lazarus and Barden, 1964; Wachstein and Besen, 1964; Wachstein and Fer nandez, 1964; Gauthier and Padykula, 1965; Farquhar and Palade, 1966; Hoff and Graf, 1966; Mao and Nakao, 1966; Re chardt and Kokko, 1967; Wills, 1967; Anderson, 1968). Many and diverse tissues have been studied concern ing the histochemical localisation of their ATPase content and speculations as to the possible or probable roles in the func tion of their respective tissues have sometimes been put for ward. A few examples from this vast area of research are pre sented. The literature cited is by no means extensive nor complete. In striated muscles there is a constant turnover of energy associated with the processes of contraction and re laxation. Many of these cellular functions require the splitt ing of ATP with the concomitant release of energy. Following this line of thinking, it is expected that ATPase activity will be demonstrable histochemically in muscular tissue. In fact, histochemical data on muscle ATPase is available (Pady kula and Herman, 1955a, 1955b; Padykula and Gauthier, 1963; Barden and Lazarus, 1964; Gauthier and Padykula, 1965; Klein, 1966; Gauthier, 1967; Grossman and Heitkamp, 1968; Brooke and Kaiser, 1969; Ogawa and Mayahara, 1969). As many as four different ATPases have been localised in skeletal muscle fibres; a mitochondrial and a myofibrillar ATPase, and two sarcotubular ATPases (Padykula and Gauthier, 1963; Gauthier and Padykula, 1965; Gauthier, 1967)..It was possible to differen tiate among these four enzymes in terms of their pH optima and their response to activators and inhibitors. Of particular in terest in the muscle are the two sites of sarcotubular activity, one at the region of the triad and the other at the region of the H band (Gauthier and Padykula, 1965; Gauthier, 1967). It is suggested that the ATPase at the triad may be involved in the accumulation of calcium ions during the period of relaxa tion, as well as in the release of calcium ions following a stimulus for muscular contraction. The ATPase at the H band is more specifically associated with the rebinding of calcium ions during relaxation (Gauthier and Padykula, 1965; Gauthier, 1967). The liver is an important and a functionally active organ whose diverse activities contribute to the structural and functional stability of the whole organism. Morphologically it is a simple organ but metabolically it is not so. Many in vestigators have applied the histochemical methods for the loc alisation of ATPase to the liver (Padykula and Herman, 1955a, 1955b; Essner et al., 1958; Novikoff et al., 1958; Holt and Hicks, 1961; Novikoff et al., 1961; Persijn et al., 1961; .Wa-ah-stain and Bradshaw, 1962; Ashworth et al., 1963; Wachstein and Fernandez, 1964; Moses et al., 1966; Wills, 1967). The products of the enzymatic reaction have been found in the membranes of the endoplasmic reticulum (Wachstein and Fernandez, 1964) but more commonly on the membranes of the microvilli of the bile canaliculi (Essner et al., 1958). The possible participation of these ATPases in molecular transport and/or pinocytosis is suggested (Essner et al., 1958). From biochemical studies it was found that nervous tissues possess a large amount of the enzyme ATPase (Bonting et al., 1962). However, biochemical data is only statistical and does not indicate the sites of enzymatic activity. Histo-chemical methods are therefore employed to localise the ATPase activity in various components of the nervous system (Novikoff et al., 1961; Tewari and Bourne, 1963a, 1963b; Torack and Barr-nett, 1963; Torack and Markey, 1964; Rechardt and Kokko, 1967). Torack and Barrnett (1963) found reaction product associated with plasma membranes of neurons and neuroglial dendrites ad jacent to the cell body, synaptic terminals and glial foot pro cesses adjacent to capillary walls. They put forward the idea that the ATPase present on the membranes of the glial processes may be related to the supposed function of the astrocytes in transporting materials between blood vessels and neurons. At the synapses the enzyme may participate in the synthesis of acetylcholine. Interestingly enough, there is no enzymatic act ivity in the endothelial cells of the cerebral capillaries, that is, those of the blood-brain barrier, while enzymatic act ivity is present in other endothelia. Involvement in transfer mechanisms across the walls of small blood vessels is implied. Water, salts and nutrients cross the capillary endothelium to the surrounding cells by active transport and pinocytosis. Simultaneously, waste products from the cells are removed in the blood by similar processes. Probably the enzyme ATPase is necessary for these processes to occur. Perhaps one of the causes for the failure of an interchange of materials between the en dothelial cells of the cerebral capillaries and the brain cells, -is the lack of the enzyme ATPase leading to an inability of the endothelial cells to carry out transport processes. ATPase activity has been localised histochemically in practically every system in the vertebrate body (Novikoff et al., 1961; Wachstein and Bradshaw, 1962; Ashworth et al., 1963; Barden and Lazarus, 1963; Bradshaw et al., 1963; Otero-Vilardebo et al., 1963; Goldfischer et al., 1964; Lazarus and Barden, 1964; Wachstein and Fernandez, 1964; Wheeler and Whi-ttam, 1964; Essner et al., 1965; Farquhar and Palade, 1966; Hoff and Graf, 1966; Mao and Nakao, 1966; Tormey, 1966; Yeth-amany and Lazarus, 1967; Anderson, 1968; Marchesi, 1968; Abel, 1969). The urinary system, with particular emphasis om' the kidney, is no exception (Padykula and Herman, 1955b; Spater et al., 1958; Freiman and Kaplan, 1959; Kaplan and Novikoff, 1959; Freiman and Kaplan, I960; Novikoff et al., 1961; Persijn et al., 1961; Pinkstaff et al., 1962; Wachstein and Bradshaw, 1962; Ashworth et al., 1963; Otero-Vilardebo et al., 1963; Wachstein and Besen, 1963, 1964; Wheeler and Whittam, 1964; Wachstein and Bradshaw, 1965; Jacobsen et al., 1967; Abel, 1969; Jacobsen and Jorgensen, 1969). Most of the research on kidney ATPase, both with the light microscope and the electron microscope, has been on adult kidneys. Few studies have been carried out on newborn or developing kidneys (Pinkstaff et al., 1962; Wachstein and Bradshaw, 1965). These have only been at the level of the light microscope. Up to date, there is no description of ATPase act ivity in newborn kidneys at the level of the electron micros cope, to the knowledge of the author. The present study is an attempt to confirm the ob servations of other investigators on the localisation of the re action product for ATPase in adult kidneys using both the lead (Wachstein and Meisel, 1957) and calcium (Padykula and Herman, 1955a, 1955b; Padykula and Gauthier, 1963) methods at the el ectron microscopical level. These methods are extended to in clude newborn kidneys. These are known to have different trans port capacity and might therefore show differences in ATPase activity, if the two processes are related. Generally, the new born kidney is functionally inefficient and cannot withstand changes in acid and base intake (Wacker et al., 1961; Moog, 1965), or changes in hydration (Pinkstaff et al., 1962; Wach stein and Bradshaw, 1965). Rates of glomerular filtration and tubular absorption are low when compared with those of the adult kidney. The modifiers of enzymatic activity, p-hydroxymercuri-benzoate (PHMB) and L-cysteine, are used in an initial effort towards determining the specificity of the enzymatic activity demonstrated. Although the specificity of the enzyme system is not fully and thoroughly investigated, it will be designated as ATPas~e in the following report. II MATERIALS AND METHODS A. MATERIALS Kidneys were obtained from adult, 3 day-old, 24 hour old, 12 hour-old, 2 hour-old and 1 hour-old rats. Adult rats were killed by an intra-peritoneal injection of sodium pento-barbitol (0.6 cc of 330 mg. sodium pentobarbitol/10 ml. water) Newborn rats were killed by decapitation. The kidneys were re moved immediately and placed, in a dish containing 5% glutaral-dehyde buffered with 0.1M sodium cacodylate (Sabatini et al., 1963). Cortical tissue was cut with sharp razor blades into 1 mm. cubes and immersed in the fixative. (All the procedures were carried out at room temp erature unless otherwise stated.) B. PRE-EIXATION IN GLUTARALDEHYDE To combine histochemistry with electron microscopy, it was found that pre-fixation was often necessary to adequate ly preserve the ultrastructural features of the tissue (Barr-nett, 1959; Holt and Hicks, 1961). Without pre-fixation there was a loss of fine details and therefore it was difficult to determine with which structures the products of the histochem ical reactions were associated. Osmium tetroxide, the most common and useful fixative then, gave excellent cytological fixation but seriously reduced or destroyed the activity of many enzymes (Holt and Hicks, 1961; Sabatini et al., 1963). There was need for a fixative which would retain satisfactory ultrastructure as well as preserve enough enzymatic activity to be demonstrable with histochemical techniques. Buffered formaldehyde was used by. Holt and Hicks (1961). Sabatini et al., (1963, 1964) introduced glutaraldehyde and other di-aldehydes as suitable fixatives for ultrastructural and cyto-chemical studies. Glutaraldehyde is the most widely used at the present time. 8. Commercial glutaraldehyde preparations are rather crude and have to be purified by various means (Fahimi and Drochmans, 1968). In these experiments, the stock solution of 25?& glutaraldehyde (Eastman Organic Chemicals, Distillation Products Industries, Rochester 3, New York) was always filter ed through activated charcoal (Fahimi and Drochmans, 1968) just prior to use. As will be discussed later, the duration of pre-fixation in 57° glutaraldehyde-cacodylate (0.1M) affected ATPase activity at pH 9.4 especially in the kidneys of newborn rats. The times of fixation were varied as indicated in Table I. TABLE I AGE OF THE ANIMALS AND THE LENGTH QF FIXATION OF THE RESPECTIVE KIDNEYS Age of animals Length of fixation (Hours) Adult 5 3 3 days 5 24 hours 5 3 12 hours 4 2 2 hours 2 1 hour 14- i 4- i Following fixation in glutaraldehyde, the tissue blocks were washed in the cacodylate buffer for at least 1 hour or stored in buffer overnight (4° C) prior to incubation. C. INCUBATION FOR HISTOCHEMISTRY Two general procedures were followed for the demon stration of ATPase activity in the kidney. At pH 7.2 the lead method (Wachstein and Meisel, 1957) was used while at pH 9.4 the calcium method (Padykula and Herman, 1955a, 1955b; Pady kula and Gauthier, 1963) was used. (a) Preparation of incubating medium at pH 7.2 12.ml. distilled water 20 ml. Tris maleate buffer (pH 7.2) 3 ml. 2$ lead nitrate (It was added gradually with constant stirring to avoid precipitation.) 5 ml. 0.1 M magnesium sulfate 25 mg. ATP (Sigma) The pH of the solution was adjusted to 7.2 if necessary. It was filtered and made up to 50 ml. (b) Preparation of incubating medium at pH 9.4 10 ml. 2#> sodium barbitol 5 ml. 2fo calcium chloride 15 ml. distilled water 0.15 mg. ATP (Sigma) was added to the above mixture, the pH adjusted to 9.4, filtered and filled up to 50 ml. with distilled water. The solutions were always made up fresh and used immediately. Routinely, the tissue blocks were incubated in the respective media at 37°C for 2 hours. Following incubation at pH 7.2, the tissue was rinsed with and stored in buffer over night at 4°C. Following incubation at pH 9.4, the tissue was washed in several changes of ifo calcium chloride for \ hour and several changes of 2% cobalt nitrate for 15 mins. before being kept in buffer overnight at 4°C. 10. D. EFFECT OF MODIFIERS The response to modifiers (activators and inhibitors) was tested using 2.5 x 10*"^ M! L-cysteine (Sigma) as a source of sulfhydryl groups, and 2.5 x 10"^ M PHMB (Sigma) as a mer curial compound (Padykula and Herman, 1955b; Gauthier, 1967). PHMB is a mercaptide forming agent and inhibits enzymes having sulfhydryl groups at their active centres (Glenner, 1968). L-cysteine, an SH- compound, would activate an SH-dependent enzyme, while at the same time strongly inhibit any alkaline phosphatase activity (Padykula and Herman, 1955b). It is be lieved that alkaline phosphatase could act on ATP as a subst rate (Padykula and Herman, 1955b; Freiman and Kaplan, 1959; Persijn et al., 1961; Hori and Chang, 1963). It has been shown that any modifier should be used both before and during incubation to overcome the effects of "differential diffusion of modifier and substrate" (Glenner, 1968). A pre-incubation exposure was effected by adding either L-cysteine or PHMB to the glutaraldehyde-cacodylate fixative for the last hour of fixation. For comparison some tissue blocks were not pre-incubated with the modifiers in the fixate ive. PHMB is highly insoluble if it is added directly to the incubating media, as has been suggested (Gauthier, 1967).' Therefore it was first dissolved in a dilute alkaline solution of sodium hydroxide before being incorporated into the incub ating media or the fixative. The pH's of the respective solutions were then checked and adjusted. E. CONTROLS Control specimens were run simultaneously with the experimental specimens. In the control preparations, all the above procedures were followed with the exception that ATP was omitted from the incubation mixtures. 11. F. POST-FIXATION IN OSMIUM The tissue was post-fixed, following incubation and storage overnight in buffer, for 1 hour in 1$ osmium tetroxide buffered with 0.1 M sodium cacodylate. This increased the con trast of the membrane systems and stabilised the fine struct ure of the cells maintained by glutaraldehyde for Epon embedd ing (Sabatini et al., 1963, 1964). G. ELECTRON MICROSOPPY The tissue blocks were dehydrated through a graded series of alcohol, half and half of absolute alcohol and propy lene oxide, and propylene oxide alone. The tissue was then in filtrated with a 1:1 mixture of propylene oxide and Epon for • 2 to 3 hours. Each tissue block was embedded in a gelatin cap sule containing fresh Epon, and the Epon allowed to polymerise overnight in a 65°C oven. Sections were cut with glass knives on a Porter Blum MT-2 ultramicrotome. Silver or gold sections were picked up on uncoated copper grids and examinedri^ri'th a Philips EM 200. All the sections were unstained. 12. Ill OBSERVATIONS A. MORPHOLOGICAL BASIS FOR HISTOCHEMICAL INTERPRETATIONS (a) Adult kidney The structure of the adult kidney has been studied extensively by morphologists and anatomists with the naked eye and under the light microscope (Maunsbach, 1966a, 1966b; Tisher et al., 1966; Rouiller, 1969 - a review). V/ith the introduct ion of the electron microscope, some of the older and class- -ical descriptions of renal structure have been confirmed and -also much extended (Pease, 1955; Yamada, 1955; Suzuki, 1958; Maunsbach et al., 1962; Porter and Bonneville, 1964; Bulger, 1965; Maunsbach, 1966a, 1966b; Tisher et al., 1966; Griffith et al., 1967; Latta et al., 1967; Ericsson and Trump, 1969; Simon and Chatelanat, 1969). Only a brief description of the more prominent ultrastructural features of the proximal tubule, dist al tubule and glomerulus will be presented here. These charact eristics were the criteria used for assigning the enzymatic re action for ATPase to a given portion of the nephron. To the naked eye, the kidney which has been freshly removed, is a glistening, reddish-coloured, bean-shaped struct ure. It is firm to the touch. A section through the kidney reveals a clear demarcation between the reddish-brown cortex -and the paler medulla. Only the cortical structures were studied in this present investigation. The proximal tubule (Pease, 1955; Porter and Bonn eville, 1964; Bulger, 1965; Trump and Ericsson, 1965; Mauns bach, 1966a, 1966b; Tisher et al., 1966; Latta et al., 1967; Ericsson and Trump, 1969) constitutes most of the cortex. The epithelium of the tubule consists of a single layer of trun cated pyramidal cells. In the apical portion of the cells bordering the lumen, the plasma membrane is thrown into folds to form numerous closely packed microvilli. These constitute the brush border (Figures 1, 18-19, 22, 24, 37, 40). An 13. electron-opaque, PAS positive layer of material, the glyco-calyx, covers the plasma membrane of the microvilli (Trump and Ericsson,,1965; Latta et al., 1967; Ericsson and Trump, 1969). Within the microvilli, a core of electron-dense-mater ial is- sometimes present. (Figure 19) Small tubular invaginat-ionssfrom the bases of the microvilli, vesicles and vacuoles of various sizes, are abundant in the apical cytoplasm (Figures 1, 18-19, 22, 24, 40). It is postulated that these invaginat ions, vesicles and vacuoles represent one pathway for tubular reabsorption of larger molecules (Porter and Bonneville, 1964; Latta et al., 1967; Ericsson and Trump, 1969). In the basilar zone of the proximal tubule cells, deep infoldings of the plasma membrane divide the cytoplasm in to numerous slender compartments within which mitochondria are enclosed. The processes from one cell interdigitate extensive ly with processes from adjacent cells (Figures 2, 18, 22-23, 40). As expected of these functionally active cells, a great number of mitochondria, closely associated with the infolded membranes of the basilar processes, are present. These mitoch ondria are large, elongated and possess numerous cristae. They are oriented perpendicular to the basement membrane (Figures 1, 23). At the lateral cell surface, there is also extensive inter-digitating cytoplasmic processes. Some are small and are con fined to the apical or basilar regions of the cell, whereas others extend from the apex to the base (Bulger, 1965). The brush border, basal and lateral interdigitations greatly in crease the surface area of the cells and therefore, also the number of sites where enzymatic reactions can occur. A nucleus, organelles and inclusions are present within the cytoplasm. The basement membrane forms a continuous layer around the proximal tubule. The epithelium of the distal tubule (Latta et al., , 1967; Ericsson and Trump, 1969).is lower than that of the proximal tubule. The cells are cuboidal in shape. In the region of the nucleus, the cells may bulge into the lumen. The apical plasma membrane is not so highly differentiated structurally 14. as in the proximal tubule. There is no distinct brush border although frequently there are some small, short microvilli (Fig ure 38). A large number of vesicles may be seen in the apical cytoplasm. A prominent characteristic are the numerous basilar processes containing large and slender mitochondria, which may extend almost to the luminal surface (Figures 3, 38). The fine structure of the glomerulus as seen under -the electron microscope was described as early as 1953 by Hall. Since then there have been many more studies on the ultrastruct-ure of the normal (Pease, 1955; Yamada, 1955; Hall, 1957; Porter and Bonneville, 1964; Jones, 1969; Simon and Chatelanat, 1969) and pathological (Simon and Chatelanat, 1969) glomerulus. The three components of the glomerulus are the base ment membrane, bounded on one side by the capillary endothelium and on thenother by the visceral epithelium of Bowman's capsule (Figures 4, 20-21, 25, 39). The cytoplasm of the endothelium is extremely attenuated. These cytoplasmic prolongations possess round pores or fenestrae. It is only in the region of the nucl eus that the endothelial cell projects into the capillary lumen. The basement membrane, interposed between the endo thelium and the epithelium, forms, a continuous barrier in the filtration process. It is composed of three layers, the lamina rara externa, the lamina densa and the lamina rara interna. The dimensions of the three layers vary with the species. In the adult rat the lamina densa is quite thick and prominent (Fig- : ures 4, 21, 25, 39). The visceral epithelial cells of Bowman's capsule, or better known as the podocytes, are highly specialised. The cells send out small cytoplasmic prolongations, the foot pro cesses, which are apposed on the lamina rara externa of the -basement membrane. The foot processes from one podocytic cell interdigitate extensively with those of adjacent cells thus giving rise to an intricate network. The foot processes from one podocyte may rest on the basement membrane of several capillaries (Figure 4). Conversely, each capillary may receive contributions from more than one podocytic cell. Recently, it 15. has "been demonstrated that a coat of neutral and acid muco-substances invests the plasma membranes of the foot processes as well as the podocytic cell bodies (Jones, 1969). (b) Newborn kidney The fresh, unfixed kidney of a newborn rat is small, pale and translucent. It is soft to the touch and its * shape is easily deformed by applying pressure. During the first few weeks of life, the rat kidney grows through the formation of new nephrons in the nephrogenic zone of the cortex. Its.?' weight in creases seven-fold within the first ten days (Wachstein and Brad shaw, 1965). In sections of the kidney cortex, the tubules and glomeruli are not closely packed together as in the adult kid ney. Instead an abundant stroma of mesenchymal elements sep arates the tubules from each other and from the glomeruli. Tub ules in various stages of differentiation are always present within the same specimen at any one time (Clark, 1957; Suzuki,' 1958). This was found to be so even in a 1 hour-old kidney. Some cortical tubules appear relatively undifferent iated (Figures 5-7, 11-12, 26-28, 34-35, 41). The cells are low and a large nucleus occupies most of the cell volume. The cyto plasm looks simple under the electron microscope. There are few organelles or membranous structures, with the exception of some small, round mitochondria randomly distributed throughout the cytoplasm. The mitochondria possess only a few cristae. The plasma membrane at the luminal surface is simple in contour or may be folded to form some short microvilli (Figures 6-7, 11-12, 27-28). An occasional cilium is present (Figure 35). The basilar membrane too, is not as complex as in the adult with -few, if any, interdigitations. The lateral membrane between two adjacent cells is simple, or the beginnings of cytoplasmic interdigitations may be observed (Figures 5, 26-27). At this stage, it does not seem possible to distinguish between the various types of tubules characteristic of each segment of the nephron. 16. In the process of renal cellular differentiation, the simple and primitive epithelial cells are transformed into highly differentiated functional cells. The successive stages -of this process has not been fully worked out yet (Du Bois, 1969). An essential component of renal cellular differentiate ion is the elaboration of cell membrane for the formation of the brush border in the proximal tubules, and the basal and lateral interdigitations in both the proximal and distal tubules. Among the differentiating tubules, the developing proximal tubule (Figures 8, 13-15, 30-32, 41, 44) is most easily ident ifiable by the presence of long, slender microvilli which may still be sparse or may be closely packed together. The brush border is acquired through the progressive accumulation of ap ical microvilli (Clark, 1957; Suzuki, 1958; Du Bois, 1969). It was observed that where a brush border was prominent, apical vesicles and vacuoles as well as a number of electron-dense granules were also present (Figures 8, 14-15, 30-32). There is an increase in the number of basal and lateral cytoplasmic inter digitations (Figures 8, 31-32). Simultaneously the mitochondria become regularly aligned perpendicular to the basement membrane within the basilar cytoplasmic compartments. Clark (1957) views the formation of the basilar infoldings as a result of progressive flutings of the cell membrane. Suzuki (1958) how ever, hypothesises that vesicles gather around the basilar portions of the cells and around the mitochondria. The vesicles coalesce to form small, flattened sacs which in turn come to gether as larger sacs wrapping around the mitochondria. By this process, the basilar cytoplasmic compartments are formed. As with the cortical tubules, glomeruli in many stages of development are present within the same specimen (Figures 9-10, 16-17, 33, 36, 43). Some glomeruli are struct-' urally immature (Figures 9, 16-17, 36)while others have the form of adult glomeruli although smaller in size (Figures 10, 33, 43). Du Bois (1969) describes the progressive stages of development of the glomerulus in the embryonic kidney. In its earliest form, a mass of podocytic cells, prismatic in shape 17. (Figures 9, 16, 36), denotes the region of the glomerulus. Further along in development, the apical pole of the podocytic cell containing the nucleus bulges out into Bowman's space, while the basal pole sends out cytoplasmic prolongations which differentiate into the foot processes (Figures 17, 33, 43). These increase in length and complexity and come to lie on the trilaminar basement membrane separating the podocytes from the endothelium. Concomitant with the development of the foot pro cesses, the endothelial cytoplasm becomes attenuated and fen estrated, thus increasing the diameter of the capillaries (Eigure 10). B. EFFECT OF FIXATION ON ULTRASTRUCTURB (a) Adult kidney As early as 1955, Pease observed that small diff erences in the preparation techniques when applied to the kid ney, could cause, large variations in the morphology of the kidney tubular elements. Since then his observations have been substantiated (Maunsbach et al., 1962; Trump and Ericsson, 1965; "Maunsbach, 1966a). The apical ends of the cells are especially labile. The proximal tubule cells show great sensitivity to fixation conditions and are affected both by the character of the fixative and by the method of application of the fixative solution. In the present study, cortical tissue from the ex cised kidney was fixed by the immersion of small blocks in the fixative solution consisting of 5f° glutaraldehyde buffered with 0.1 M sodium cacodylate. In general, the fixative used gave adequate preservation of the organelles in the cytoplasm. However, by this method of immersion in the fixative, some •artifacts are present in the proximal tubules (Maunsbach et al., 1962; Maunsbach, 1966a; Tisher et al., 1966). The lumens •of the proximal tubules were more often than not closed resulting in a region of closely packed microvilli (Figures 1, 18-19, 22, 37). In vivo, the lumens are found to be open 18. with a regular brush border, so that such collapsed tubules were artifactual resulting from excessive swelling of the cells during fixation (Maunsbach, 1966a). There may be some cell ular debris in the lumens of the tubules. These take the form of cytoplasmic bits and pieces, or even whole, less osmiophilic cells that seem to be extruded into the lumen. An occasional tubule was not collapsed but possessed a patent lumen. Tisher et al. (1966) interprets this as due to dehydration in the preparative techniques for electron microscopy following cell swelling during fixation. Ocaasionally the membranes in the basilar part of the cells were separated giving rise to extra cellular compartments of different sizes. The extent of the presence of extracellular compartments was not consistent (Fig ures 2-3, 18, 22-23, 38, 40). Variations between cells in the same tubule as well as variations between different tubules were observed. These extracellular compartments are probably indications of sensitive cellular reactions to physiological and pathological (in this case, fixation) changes in the en vironment . The distal tubules and the glomeruli were more resistant to the effects of fixation and were always morphol- T ogically well preserved. The lumens of the distal tubules were open. There could have been some cell swelling! resulting in a reduction in size of the lumen but this was not so obvious as in the proximal tubules where the brush border accentuated the effects. (b) Newborn kidney The same concentration of fixative and a similar method of fixation, that is, by the immersion of small tissue blocks, was applied to the newborn kidney. The effects of fixation varied depending on the stage of differentiation of the tubules. The morphologically undifferentiated cortical tubular cells consisted of a nucleus, some cytoplasm and a few organelles, primarily small, round mitochondria with 19. sparse cristae, scattered throughout the cytoplasm. The mito chondria in the immature cells seemed more susceptible to -fixation artifacts as compared with the adult. Quite a number of the mitochondria were "exploded". (Figures 27-28, 30-31, 38) Otherwise, the preservation of ultrastructure was generally good. The1tubules had wide open lumens often filled with cell ular debris. Debris was also found in the extratubular stroma. There appeared to be more cellular debris associated with the newborn kidney than with the adult kidney. In cross-sections of some undifferentiated tubules, all the cells but one, were well preserved. This one cell was completely disintegrated with a nucleus that was swollen to immense proportions (Figure 29). Such a phenomenon was not observed in adult kidney tissue. Developing proximal tubules were recognised by the presence of long, slender microvilli in the apical surface membrane (Figures 8, 13-15, 30-32, 38). They may be few in number or may be closely packed to form a distinct brush border. In the tubules with few microvilli, the lumens were open but contained some cellular debris. In more advanced proximal tub ules, that is, those having a well-developed brush border and a number of vesicles and vacuoles in the apical cytoplasm, the lumens were closed (Figures 8, 15, 31-32). Less osmiophilic cells were often seen being extruded into the lumen (Figure 8). The response of these tubules to fixation was very similar to that of the adult. The mitochondria were generally still small and showed some artifacts of fixation. The glomeruli were always adequately preserved ultra -structurally independent of the stage of development. G. EFFECTS OF FIXATION ON ENZYMATIC ACTIVITY The histochemical localisation of ATPase enzymatic activity has most often been carried out on the adult kidney using the lead method at pH 7.2 proposed by Wachstein and Meisel (1957). Those investigators who used both the lead method at pH 7.2 (Wachstein and Meisel, 1957) and the calcium method at pH 9.4 (Padykula and Herman, 1955a, 1955b; Padykula and Gauth-20. TABLE II THE EFFECT OF FIXATION ON ENZYMATIC ACTIVITY Age of animals Time of fixation (hours) Lead method pH 7.2 Calcium method pH 9.4 P d g P d g Adult 5 + + + + + + Adult 3 + + + 3 days 5 + + + + + -24 hours 5 + + + - - -24 hours 3 + + + 12 hours 4 + - -12 hours 2 + + + 2 hours 2 + + + + - -1 hour IT - - -1 hour 1 - - -1 hour JL 2 - - -1 hour JL 4 + + + Fixative; 5f° glutaraldehyde in 0.1M sodium cacodylate buffer at pH 7.2. + reaction precipitate present reaction precipitate absent p proximal tubules d distal tubules g glomeruli -NB. In newborn tissue, wherever it was not possible to differ entiate between a developing proximal tubule and a develop ing distal tubule, it was assumed that the undifferentiated tubules represented both types. 21. ier, 1963) reported that>similar results were obtained with either method (Novikoff et al., 1961). In the adult kidney, these observations on plasma membrane ATPase activity was found to be so. However, with immature newborn kidneys it was ob served that the length of fixation affected the localisation of membrane ATPase activity differently depending on the method that was employed (See Table II). The time of fixation was not important when deal ing with adult kidney tissue. Even up to 5 hours fixation time there was much reaction precipitate associated with the membranes, using either the lead method at pH 7.2, or the calcium method at pH 9.4. The time of fixation however, was more critical in the case of immature newborn kidneys, if the localisat ion for enzymatic activity was carried out at pH 9.4. A 2 hour-old kidney after 2 hours in the fixative solution still showed reaction product for the enzyme reaction at pH 7.2. But little or no precipitate was present at pH 9.4 with a similar time of fixation. It seemed that the more immature the kidney in terms of age postnatally, the shorter the time of fixation needed to preserve enough enzymatic activity to be demonst rable at pH 9.4 with the calcium method. Adequate amounts of enzymatic activity were always present to result in a positive reaction at pH 7.2. With the kidney of a 1 hour-old rat, even a § hour of fixation was sufficient to inhibit any enzymatic activity at pH 9.4 that might have been present in the tissue. With the fixative used (5$ glutaraldehyde in 0.1M sodium cacodylate) good preservation of ultrastructure had to be sacrificed to prevent complete inhibition of any ATP-hydrolysing enzymes at pH 9.4. D. ENZYMATIC ACTIVITY OF THE ADULT KIDNEY (a) T?H 7.2 - lead method Under the electron beam, the final product of the ATPase enzymatic reaction was visualised as electron-dense 22. particles, which may be in the form of fine granules or in the form of larger aggregates. The sites of deposition of the reaction product would indicate areas of enzymatic activity. The proximal tubules showed two distinct regions of enzymatic activity, the microvilli of the brush border and the interdigitations of the basal and lateral membranes (Figures 1-2). The precipitate was most often located on the outer sur face of the plasma membranes of the microvilli. At times, but rather rarely, precipitate was associated with the inner cyto plasmic surfaces of the microvilli membranes. Precipitate could sometimes be demonstrated within some of the tubular invagin ations arising from the bases of the microvilli, and within some of the small apical vesicles and large apical vacuoles. Some of the membrane interdigitations showed such an abundant deposition of reaction product that the space between the membranes was completely filled with precipitate. Where the reaction was less intense, the precipitate was found to be on the membrane itself and not free in the extracellular space (Figure 2). However, the reaction precipitate may be found on the cytoplasmic aspect of the membranes as well as within the cytoplasm (Figure l). The basement membrane of some proximal tubules accumulated precipitate (Figure 2). Variations in staining intensity of the cells within the same tubule, as well as between different tubules was encountered. At times two tubules adjacent to each other, reacted differently. One tubule showed reaction precipitate in the brush border and basilar infoldings while the other showed little or no react ion precipitate. In the distal tubules the reaction product was confined mainly to the cell membranes of the basilar inter-digitating cytoplasmic processes and the lateral membranes separating two adjacent cells (Figure 3). As in the case of the proximal tubules, precipitate may be found on both the extracellular and cytoplasmic aspects of the interdigitating membranes and within the cytoplasm (Figure 3). Occasionally, the few short microvilli reacted positively and precipitate 23. was seen on the microvilli membranes. The glomeruli were sites of active enzymatic activ ity (Figure 4). Abundant precipitate was present both on the membranes and within the cytoplasm of the foot processes, in the trilaminar, basement membrane but not in the endothelium (Figure 4). (h) pH 9.4 - calcium method The results from the localisation of ATPase act ivity with the calcium method at pH 9.4 were similar to those obtained with the lead method at pH 7.2. Enzymatic activity was demonstrated in the brush border of the proximal tubules, in the lateral and basal interdigitations of the proximal and distal tubules, and in the glomerular epithelial cells. E. ENZYMATIC ACTIVITY OF THE NEWBORN KIDNEY (a) pH 7.2 - lead method (Figures 5-10) With this method it was possible to localise enzy matic activity in the kidneys of newborn rats of various ages, ranging from a 2 hqur-old to a 3.day-old. The time of pre-fixation in ~fo glutaraldehyde-cacodylate did not appear to affect the level of enzymatic activity. In a very immature kidney, for example from a 2 hour-old rat, most of the tubules were still undifferentiated and it was not possible to distinguish between a proximal tubule and a distal tubule (Figures 5-7). A large number of these undifferentiated tubules reacted intensely when incubated in the lead-ATP medium. The lateral membranes between neighbour ing cells, which were more often simple in contour, were fill ed with the reaction product. In fact, the boundaries between individual cells in the tubules were clearly outlined by the deposition of the precipitate in the lateral membranes (Fig- • ures 5-7). Some precipitate was found on the plasma membranes lining the lumens of the tubules (Figure 6). Some of the microvilli that were present reacted positively while others reacted negatively in the incubating medium. At this early 24. stage of differentiation, the basal membranes did not show-any elaborate infoldings as seen in the adult kidney (Figures 5-7). There was usually no precipitate on the basal membranes (Figures 5-6). Sometimes, however, a single cell within a tubule showed abundant precipitate on the basal membranes (Figures 5, 7). This was an exception. Besides the undifferentiated tubules, tubules in various stages of differentiation were also present within the newborn kidney. A developing proximal tubule possessed a fair ly well-developed brush border and many more interdigitations of the basal and lateral membranes (Figure 8). These inter digitations were still by no means as extensive as in the adult. Reaction precipitate was found encrusting the plasma membranes of the microvilli in the brush border (Figure 8). Occasionally some precipitate was present within the micro villi. The basal infoldings had fine precipitate adhering to the, membranes. Usually the space between the membranes was not filled up with precipitate as was the case with the un differentiated tubules (Figure 8). , . . Developing distal tubules showed an increase in the number of short, irregularly shaped microvilli. The re action precipitate generally coated the membranes of the micro villi but was also seen within the cytoplasm of the microvilli. The lateral and basal interdigitations were frequently so packed with precipitate that it was impossible to tell if the precipitate was just confined to the outer surface of the membranes, or was also present on the inner cytoplasmic sur face as well. As with the tubular elements in the kidney, im mature undifferentiated glomeruli were present together with more well-developed glomeruli within the same specimen. In the undifferentiated glomerulus, the podocytic cells were closely packed so that their limiting membranes were quite often in apposition (Figure 9). Some podocytes showed the beginnings of cytoplasmic prolongations which would eventually develop to form the foot processes. It was observed that re-25. action product was almost always present where two sets of membranes were apposed.(Figure 9). Portions of the membranes of the podocytic cell which was not close to the membranes of another podocytic cell sometimes showed precipitate but this was not usually so. Not all the cells in the same glom erulus were reactive. Some cells showed no detectable enzy matic activity. In a glomerulus further along the course of differ entiation, the foot processes of the podocytic cells had in creased in number and formed a complex network along the; tri laminar basement membrane.. The cytoplasm of the endothelial cell was becoming attenuated and fenestrated but not to sucM a great extent as in the adult (Figure 10) Reaction precipit ate was found on the membranes as well as within the cyto plasm of the foot processes and podocytic cell body, which was still situated quite near to the capillaries. The endo thelial cells were usually non-reactive. If precipitate were present, it was found mainly on the cytoplasmic surface of the membranes and in the cytoplasm (Figure 10). (b) pH 9.4 - calcium method (Figures 11-17) FormATPase enzymatic activity in a newborn kidney to be demonstrable with this method, the time of pre-fixation in glutaraldehyde was found to be critical (Refer to Table II). A 3 day-old kidney fixed for 5 hours prior to in cubation showed enzymatic activity in the proximal and distal tubules but not in the glomerulus. In the proximal tubule, precipitate was found all along the infoldings and also coat ing the microvilli. Quite a number of the apical tubular in vaginations from the bases of the microvilli were lined with the fine precipitate (Figure 15). The lateral'and basal inter digitations of the distal tubule gave positive results. A 24 hour-aid kidney pre-fixed for 5 hours showed no reaction precipitate. But if the time of fixation was shortened to 3 hours, there were indications of enzymatic activity in the tubules. In the glomerulus, reaction product 26. was sometimes present and sometimes not. If present, it-was localised on the plasma membranes of the podocytic processes. Thus by controlling the times of fixation it was possible to localise ATPase' enzymatic activity in the tubules and glomeruli of the newborn kidney. In cases where there was just minimal activity, for example, a 12 hour-old kidney with 4 hours pre-fixation, or a 2 hour-old kidney with 2 hours pre-fixation, or sometimes a 1 hour-old kidney with 1 hour pre-fixation, reaction precipitate was observed mostly in the tubular elements. The precipitate, in the form of discrete el ectron-dense particles, were found on the membranes of the few microvilli and sometimes on the lateral membranes (Figures 11-12). Where the reaction was more intense, for example, a 12 hour-old kidney with 2 hours pre-fixation, or a 1 hour-old kidney with \ hour pre-fixation, the sites for the deposition of the reaction product for ATPase at pH 9.4 were similar to those at pH 7.2 (Figures 13-14). Some tubules reacted positive ly while others reacted negatively. This was not dependent on the degree of maturation of the tubules. In the immature tub ules, the precipitate in the lateral membranes clearly separ ated one cell from the next. Some precipitate was also present on the relatively simple plasma membrane lining the luminal surface. The brush border of the developing proximal tubules showed varying amounts of precipitate. Some of the apical vesicles and vacuoles had reaction product- on their limiting membranes (Figures 14-15). In developing distal tubules, re action precipitate was observed mainly in the lateral memb ranes even though some basal infoldings and some microvilli were present. Some of the tubules showed precipitate in -the region of the basement membrane but this did not appear to be associated with the basal membranes. Observations on the enzymatic activity of the glomerulus at pH 9.4 were similar to those at pH 7.2 (Figures 16-17). Glomerular enzymatic-activity appeared to be moressusceptible to fixation effects. With longer fixation times, enzymatic activity in the glom erulus was inhibited the most. 27. F. EFFECT OF THE MODIFIERS, PHMB AND L-CYSTEINE OH ENZYMATIC  ACTIVITY (a) Adult kidney TABLE III THE EFFECT OF THE MODIFIERS, PHMB AND L- CYSTEINE ON ENZYMATIC ACTIVITY OF THE  ADULT KIDNEY PHMB L-cysteine No Yes No Yes P d g P d g P d g P d . g pH 7.2 + - + + - + + + + + + + pH 9.4 + - + + - - + + + + - -+ reaction precipitate present - reaction precipitate absent p proximal tubules d. distal tubules g glomeruli No no pre-incubation exposure Yes with pre-incubation exposure At pH 7.2, with or without a pre-incubation ex posure to PHMB, enzymatic activity was localised mainly at the proximal tubules (Figures 18-19) and glomeruli (Figures 20-21). Within the proximal tubules, the precipitate was 28. present in the brush border as well as in the 'interdigitat ions (Figure 18). In the brush border the precipitate was us- • ually found uniformly distributed over the membranes of all the microvilli, but were at times clumped together in certain regions within the brush border (Figure 19). This was inter preted as an artifact rather than as an indication of hetero geneity of sensitivity to the effects of PHMB among the 'micro villi. Variations in the intensity of reaction at the infold -ings occured between individual cells and between different tubules (Figure 18). Only the membranes of the podocytic foot processes showed an accumulation of the reaction prod uct. There was no precipitate in the cytoplasm of these cells as was often noticed with tissue incubated without PHMB. There was also no precipitate in the basement membrane or endo thelium (Figures 20-21). At pH 9-4, enzymatic activity was demonstrable in the proximal tubules whether there was a pre-incubation expo sure to PHMB or not (Figures 22-24). In either experimental conditions, the distal tubules showed no reaction product. There was an occasional distal tubule with precipitate in the interdigitations. However, in the glomerulus a difference was ' observed between tissue that had been pre-incubated with PHMB and those that had not. Without pre-incubation, some of the glomeruli showed precipitate in the cytoplasm and on the membranes of the podocytic foot processes but not in the base ment membrane or endothelium (Figure 25). With pre-incubation, most of the glomeruli showed no reaction product. This observ ation substantiated Glenner1s (1968) suggestion that any modifier should be used both before and during incubation. This would ensure that the modifier had reached the site of enzymatic activity prior to incubation in the substrate medium. L-cysteine had no effect on the localisation of enzymatic activity at pH 7.2. But at pH 9.4, a pre-incubat ion exposure to L-cysteine inhibited practically all enzy matic activity in the glomeruli and in most of the distal tubules. 29. (b) Newborn kidney TABLE IV THE EFFECT OF THE MODIFIERS. PHMB AND L- • CYSTEINE, ON ENZYMATIC ACTIVITY OF THE NEWBORN KIDNEY PHMB L-cysteine No Yes No Yes t g t g t g t g pH 7.2 + + + + + + + + pH 9.4 - _ - - - - - -+ reaction precipitate present reaction precipitate absent t tubules g glomeruli No no pre-incubation exposure Yes "with pre-incubation exposure At pH 7.2, neither PHMB nor L-cysteine had any effects on the enzymatic activity of the differentiating tubules (Figures 26-32, 34-35) and glomeruli (Figures 33, 36). Most of the tubules showed intense reaction in the lat eral membranes between cells (Figures 26-27, 30-32, 34-35). The luminal surface membrane and microvilli, if developed, show ed variations in the amount of precipitate (Figures 30-32). 30. In developing proximal tubules, the brush border was a dist inctly active enzymatic site (Figures 30-32). The glomerulus had precipitate associated especially with membranes which were in apposition (Figures 33, 36). At pH 9«4, no enzymatic activity was detected in the newborn kidney when PELT-IB and L-cysteine were used. How ever, these negative results cannot be considered too sig nificant at the-present time. With a newborn kidney a couple of hours old, the inhibitory effects of the fixative used were found to be so overwhelming that it was impossible to assess the effects of the modifiers. G. CONTROLS In all the experiments, controls were run simult aneously with the experimental specimens. The controls differ---ed only in that ATP was omitted from the incubating media. With the calcium method at pH 9.4, no precipitate was found in either adult or newborn tissue. With the lead method at pH 7.2, there was a tendency for some precipitation in some cells (Figures 40, 44) but not in others (Figures 37-39, 41-43). In adult kidney tissue, this took the form of fine stippling of the cytoplasm, or association of the precipitate with the basement membrane or microvilli of the brush border (Figure 40). In the newborn kidney, a fine diffuse precipitate was sometimes present. If the microvilli were long and slender, as in the developing proximal tubule, there was some precipitate on the membranes (Figure 44). • These observations were not consistently present in all the specimens. If precipitate was present in the control specimens, it was always less than in the experiment al situation. H. NUCLEAR STAINING In both experimental and control specimens, nuclear staining was found to be erratic. Variations occured between 31. individual cells in the tubules and in the glomeruli. Some nuclei were perfectly free of precipitate while others show ed a large amount. Nuclei from two adjacent cells often be haved differently, with precipitate present in the one but not in the other. Wherever precipitate was observed, it was mainly associated with the nucleolus and with the denser heterochrom-atin (Figures 10, 27). Even then there were differences in intensity. Nuclear staining did not appear to follow any con sistent pattern. 32. IV DISCUSSION A. GENERAL Both the lead method at pH 7.2 (Wachstein and Meisel, 1957) and the calcium method at pH 9.4 (Padykula and Herman, 1955a, 1955b; Padykula and Gauthier, 1963) for the histo chemical localisation of ATPase activity have been used widely to demonstrate enzymatic activity in various tissues where active transport is known to occur. Recently, the specificity of the histochemical localisation of ATPase enzymatic activity with the Wachstein-Meisel procedure has been seriously quest ioned (Moses et al., 1966; Rosenthal et al., 1966; Moses and Rosenthal, 1967, 1968; Rosenthal et al., 1969). The possibility that there is non-enzymatic hydrolysis of the substrate ATP by the lead in the incubating medium was proposed. It is thought that this non-enzymatic hydrolysis of ATP by lead could account for the deposition and localisation of preci pitate on plasma membranes, the most often observed sites of intense reaction with the lead method. Moses arid Rosenthal (1968) suggest that there is "a selective affinity of certain tissue-reactive groups at the sites of localisation for the complexes formed by the interaction of lead and ATP." At the present time it is difficult to envision how this tissue factor with a selective affinity for the products of the non-enzymatic hydrolysis of ATP could account for the substrate specificities of many plasma membranes (Novikoff, 1967), the effects of modifiers only on some membranes (Novikoff, 1967) and the differing patterns of sites of localisation of precipitate obtained with the same tissue under varying conditions. Mar-chesi (1968) in his investigations of ATPase activity on red • blood cell membranes found that there was non-enzymatic hydrolysis of ATP in the incubating medium but that it did not account for the deposition of the lead phosphate on the red blood cell membranes. Jacobsen and Jorgensen (1969) found 33. the staining of the plasma membranes of the kidney character istic of enzymatic hydrolysis rather than non-enzymatic hydro lysis, while Grossman and Heitkamp (1968) found no measurable non-enzymatic hydrolysis of ATP by lead in chemical assays of the media used for the histochemical localisation of ATPase activity. We are. far from an understanding of the complexities of the reactions involved in the reaction mixtures used in histochemistry, especially where a heavy metal like lead is present. It is probable that the various constituents of the incubating media interact with each other. There is a suggest ion that lead in the incubating media may not be in the form of free ions but may form chelates (Tetas and lowenstein, 1963; Berg, 1964; Tormey, 1966; Rechardt and Kokko, 1967; Moses and Rosenthal, 1968; Tice, 1969). This would then imply that the reaction precipitate on tissue sections may not be just simple lead phosphate but may be a more complex compound (Marchesi, 1968; Moses and Rosenthal, 1968). It is essential that variable of the reaction be carefully controlled. In the present study of ATPase enzymatic activity in both the adult and newborn kidney tissue, the composition of the incubating media were kept constant so that the significance of any differences in the sites of deposition of the reaction precipitate could be assessed. Also a method not involving the use of lead salts was deemed desirable as a comparison (Goldfischer et al., 1964) Therefore both the lead and calcium methods were applied in the investigation of ATPase enzymatic activity in adult and newborn kidney tissue. Under the conditions employed in this present -study, only plasma membrane ATPase was demonstrable. Mito chondrial ATPase was not detected although there would be an occasional mitochondrion with reaction precipitate associated with it. Fixation in glutaraldehyde has been found to have an inhibitory effect on mitochondrial ATPase (Torack and Barrnett, 1963; Lazarus and Barden, 1964; Wachstein and Besen, 1964; Essner et al., 1965; Vethamany and Lazarus, 1967; Anderson, 34. 1968). Fresh unfixed tissues.are of course ideal for demon strating any ATPase activity, including mitochondrial ATPase, but this is not always possible at the level of the electron microscope. Fixation in formalin preserves mitochondrial ATP ase enzymatic activity in some tissues (Lazarus and Barden, 1962; Wachstein and Bradshaw, 1962; Ashworth et al., 1963; Bradshaw et al., 1963; Otero-Vilardebo et al., 1963; Essner et al., 1965; Lazarus and Vethamany, 1965; Rechardt and Kokko, 1967; Vethamany and Lazarus, 1967;0gawa and Mayahara, 1969) but not in others (Wachstein et al., I960; Wachstein and: Brad shaw, 1962; Barden and Lazarus, 1963; Otero-Vilardebo et al., 1963; Wachstein and Besen, 1964; G-authier, 1967). It appears that mitochondria from different tissues show different sus ceptibilities towards fixation. Where mitochondrial ATPase has been localised, it is still not fully agreed upon whether the precipitate is on the inner cristal membranes (Ashworth et al., 1963; Otero-Vilardebo et al., 1963; Anderson, 1968; Marchesi, 1968) or within the matrix (Lazarus and Barden', 1962, 1964; Lazarus and Vethamany, 1965; Rechardt and Kokko, 1967; Vethamany and Lazarus, 1967; Grossman and Heitkamp, 1968'; Ogawa and Mayahara, 1969) of the mitochondria. To further complicate the issue of mitochondrial ATPase, a recent paper suggests that mitochondria from different tissues may have different affinities for lead salts (Wilson, 1969)• There are three possible explanations to bear in mind when considering the localisation of reaction precipitate• at the plasma membranes of kidney tubular cells and glomerular cells. Firstly, fixation of the kidney tissue in glutaralde-hyde could alter the cell membranes in such a way that they act as barriers to substances entering the cells. This being the case, enzymes present within the cytoplasm and in the cell organelles would diffuse towards the membranes and react with the substrate in the incubation medium, that is ATP, thereby releasing the reaction precipitate at the extracellular aspect of the cell membranes. This seems unlikely in view of the experimental results presented here. Although reaction 35. precipitate is most frequently observed on the extracellular aspects of the plasma membranes (Figures 2, 5-9, 11-18, 20-23, 26-27, 30-36), it is.also present within the cytoplasm (Figures 1, 3, 4, 10, 25, 36), in the nuclei (Figures 10, 27) and occasionally within the mitochondria. It appears that substances from the substrate medium can enter the cells. It is entirely possible that fixation has altered the membranes in some way. Secondly, in undifferentiated tubules of the new born kidney intense reaction precipitate is present on the lateral membranes between individual cells. In the glomeruli, apposed membranes show an accumulation of reaction product. It is suggested that the enzymes on the luminal surface memb ranes and basal membranes of the tubular cells, and on the free membranes of the podocytic cells may not be so firmly attached to the membranes and are therefore "washed" towards the apposed membranes. If this were the case, then one would expect gradients in the intensity of the deposition of the reaction product..This was not observed. Occasionally, there would be intense reaction precipitate on the luminal surface membranes (Figure 27) or on the basal membranes (Figure 5) or on the free membranes of the podocytes (Figures 16, 36). Thirdly, the localisation of reaction precipitate on the membranes could indicate ATPase enzymatic activity at the membranes themselves, as suggested here. In view of the above, it seems most likely that actual plasma membrane enzy matic activity was demonstrated in the present experiments. B. PLASMA MEMBRANE ENZYMATIC ACTIVITY (a) Adult kidney With both the lead and calcium methods for the histochemical localisation of ATPase enzymatic activity, the reaction precipitate was deposited on the plasma memb ranes of the proximal and distal tubules and glomeruli. Variations in intensity of staining were present. Besides, some tubules and glomeruli showed the presence of the react-36. ion product while others did not. These similar observations had been made previously by Wachstein and Besen (1964). This variability has been interpreted as artifactual (Goldfischer et al., 1964) although other possible explanations could also • be offered. At the present stage of development of histochem ical techniques, quantitation of enzymatic activity can only be based on the density of the final reaction product deposit ed (Glenner, 1965). Therefore variations in staining intensity found in the adult kidney when incubated for an ATPase reaction would indicate various degrees of enzymatic activity. Different nephrons at any one time may be in different functional states (Caulfield and Trump, 1962). In the proximal tubules, differ ences in the intensity of the ATPase reaction could indicate differences in enzymatic activity associated with the various segments. It has been found that proximal tubules show segment ation in terms of their ultrastructure, function and histochem ical reactions (Kissane, 1961; Maunsbach, 1966b; Tisher et al., 1966; Jacobsen et al., 1967; Latta et al., 1967; Ericsson and Trump, 1969). In the present study, the differences in the deposition of the reaction product were not correlated with the various segments of the proximal tubule. The main purpose, of course, of applying histochem ical methods for the demonstration of enzymatic activity to the kidney, is not only an attempt to localise the sites of enzymatic activity in terms of the ultrastructure of the tissue, but also to try to correlate specific sites of enzymatic act ivity with the known functions of the tissue. Unfortunately for most enzymes in the kidney, including ATPase, we have no specific idea of the role they play in kidney function. Most of the suggestions that have been put forward for the roles of ATPase in kidney function are highly speculative. Such speculations, based on available data, are useful and may lead to further experiments which might help elucidate some of the complexities of kidney function. Proximal tubules show localisation of reaction product to the membranes of the brush border, the basal and 37. the lateral interdigitations. The ATPase reaction in these sites was not eliminated by the addition of L-cysteine or PHMB to the incubating media indicating that the enzyme dem onstrated was not sulfhydryl-dependent (Padykula and Herman, 1955a, 1955b). L-cysteine served the dual purpose of being a source of sulfhydryl groups as well as an inhibitor of alkal ine phosphatase, which enzyme has been found to be also pres ent in the brush border. In this investigation L-cysteine had no effect on brush border enzymatic activity, in agreement with the findings of Padykula and Herman (1955b) but not with the findings of Freiman and Kaplan (1959) where brush border'act ivity was abolished by L-cysteine. This discrepancy is probab ly due to species differences (Wachstein and Besen, 1964) in enzymatic activity of the kidney. Padykula and Herman (1955b) examined kidneys of rats, while Freiman and Kaplan (1959) used those of dogs. In all probability, both alkaline phosphatase and ATPase are present in the brush border of the rat kidneys studied here. The precise function of the ATPase associated with the brush border is not entirely clear but it is most likely involved in the transport of substances from the tubular lumens into the cells and vice versa in the process of urine formation from the glomerular filtrate. It is an energy-requiring process involving the movement of substances up an electrochemical potential gradient. This, by definition, is "active trans port" (Solomon, 1962). Wot only ATPase (Spater et al., 1958; • Wheeler and Whittam, 1964; Ericsson and Trump, 1969) but also alkaline phosphatase (Wilmer, 1944; Rosenberg and Wil-brandt, 1952; Kissane, 1961; Matthiessen, 1966) have been implicated to take part in active transport across membranes. In much of the literature on the histochemical localisation of ATPase in various tissues, there is a tendency to consider the ATPase so localised as associated only with the sodium pump (Solomon, 1962; Post and Sen, 1965). This ATPase is sodium-potassium activated and ouabain-sensitive. There is much controversy as to whether the sodium-potassium activ-38. ated ATPase is demonstrable at all with the present available • histochemical techniques (Bonting et al., 1962; McClurkin, 1964; Tormey, 1966). The present author takes a more general view as to the nature of.the ATPase or ATPases shown by the lead and calcium methods, especially with respect to the kid ney. Besides participating in the active transport of cations like sodium and potassium, it perhaps also participates in the active transport of anions like phosphate and sulfate, sugars, amino-acids and fatty acids (loewy and Siekevitz, 1963). It is not known whether there is a common mechanism underlying the active transport of all these substances and therefore involv-' ing one common ATPase, or whether a number of ATPases are work ing in concert. The latter suggestion seems more plausible. In the brush border of the proximal tubules, small molecules presumably enter the cells by active transport across the plasma membranes. Larger molecules, colloidal mat erials and proteins probably enter by way of pinocytosis. The substances which are to be absorbed stream towards the bases of the microvilli where vesicles and vacuoles of various sizes are pinched off. This form of membrane flow probably requires energy (Loewy and Siekevitz, 1963) and therefore an ATP-dephosphorylating enzyme to make the energy available. Ericsson (1965a, 1965b) in his studies of the transport and digestion of hemoglobin, found that the hemoglobin was rapidly pino-cytosed and metabolised. Droplets containing hemoglobin app eared all below the brush border. Four hours after the intra venous injection of hemoglobin, the brush border of the prox imal tubules was abolished. Ericsson postulated that the brush' border membrane was being used up for absorption droplets and that membrane renewal was not keeping pace with membrane loss. Under physiological conditions, membrane renewal and loss would be balanced. Renewal of the plasma membrane of the brush border is conceivably an energy-requiring process with an associated ATPase. The basilar and lateral interdigitating membranes tremendously increase the surface area of the cell in contact 39. with the extracellular space. There is a large number of mito chondria closely apposed to these membranes where transport processes can occur. These membranes show an intense ATPase reaction. It has been observed that the extracellular spaces widen in response to conditions of fixation and also to var ious solutions injected intravenously (Caulfield and Trump, 1962) by increased water fluxes. The ATPase in the basilar regions of the proximal tubule cells presumably transport large amounts of salts and water. Where little correlation between structure, function and enzymatic activity in the proximal tubules is possible, there is an even lesser possibility in the distal tubules. The ATPase in the distal tubules appears to be sensitive to PHMB at pH 7.2 and pH 9.4 but also sensitive to L-cysteine at pH 9.4. The significance of this is not known. The functions post ulated for the distal tubules include active transport of sodium, potassium secretion, acidification of the urine and ammonia secretion (Ericsson and Trump, 1969). A precise correlation is not possible at the present time. In the glomerulus, enzymatic activity was localised primarily on the plasma membranes of the podocytic foot proc esses at pH 7.2 and pH 9.4. Other investigators have found re action on the endothelium alone (Kaplan and Novikoff, 1959), or on both the endothelium and epithelium (Ashworth et al., 1963; Wachstein and Besen, 1964). The ATPase in the glomerulus responded to both PHMB and L-cysteine. This could just be an indication of the glomerulus' sensitivity to any foreign' substances. It was once thought that the foot processes had only a supportive function in the glomerulus and that substances once past the trilaminar basement membrane, flowed in between the filtration slits set up by the interdigitating foot processes. There is now a suggestion that transport processes do occur across the glomerular epithelial membranes especially in relation to resorption of proteins that may have filtered across the endothelial pores and basement membrane (Jones, 1969). 40. The author postulates that in the adult rat kidney, at least two types of ATPases are localised with the lead and calcium methods. One enzyme has a pH optimum at 7.2 and the other has an optimum at 9.4. These two types of enzymes show differences in response to the modifiers PHMB and L-cysteine. The enzyme with an optimum at pH 9.4 appears to be more sens itive to the effects of modifiers. Unlike the four types of ATPases in the muscle which can be spatially separated in terms of their pH optima and their response to various activators and inhibitors (Gauthier, 1967), those in the kidney are both localised at the plasma membranes. This postulate, of course, is highly speculative. (B) Newborn kidney In the rat, the newborn kidney is not only morpho logically but also functionally immature,fas compared to the adult. The newborn kidney is an inefficient homeostatic organ and. cannot tolerate, changes in acid and base intake (Wacker et al., 1961; Moog, 1965). The newborn rats cannot handle changes' in hydration (Pinkstaff et al., 1962) which is especially evident if they are overloaded with water (Wachstein and'Brad shaw, 1965). Rates of glomerular filtration, urea clearance, absorption of glucose and accumulation of vital dyes are low when compared with those of the adult (Wachstein and Bradshaw, 1965). This functional inefficiency can generally be correlated with a less than full complement of enzymes. With morphological development, the enzymes for physiological function appear and/or increase in amount. Enzyme accumulation can be taken as one aspect of functional differentiation (Pinkstaff et al., 1962). That there is morphological immaturity in the newVx- • born kidney is quite obvious from a study of a tissue sect ion from a newborn kidney. Most of the tubular elements are undifferentiated. The extent of the plasma membrane surface area available for the transport of substances from the glom erular filtrate is small. There is no brush border, or if 41. present, only in a relatively undeveloped form. The inter digitations of the basal and lateral membranes are scarce. The glomeruli too, are mostly morphologically undifferentiated. The endothelium possess few fenestrations and the glomerular epithelial cells have few foot processes. Concomitant with the observations of morphological immaturity is the observat ion that the enzyme ATPase is relatively less abundant. In the tubules, enzymatic activirfey was noted mainly on the lateral membranes in between individual cells. There was no great accumulation of reaction precipitate on the basal membranes or luminal surface plasma membranes, undifferentiated morpho logically though they may be, as in the adult. Only with the differentiation of these membranes, as in a developing prox imal tubule, is there accumulation of reaction product, and therefore an indication of increased enzymatic activity. The weak reaction for ATPase enzymatic activity on the luminal surface membrane and few microvilli of the undifferentiated tubules would indicate some transport of substances from the tubular lumens into the cells. The shortest possible route for substances out into the extracellular space would be via the lateral membranes, where thereis intense reaction for ATPase enzymatic activity. These observations do not agree with those of Wachstein and Bradshaw (1965) who found no tubular ATPase in the newborn kidney, even in the more mature_developin_r_>prox-imal tubules. However, Pinkstaff et al. (1962) showed ATPase activity in all fetal and post-natal stages. The endothelium in the immature glomerulus often showed deposition of reaction product. Possibly active transport occurs across the endo thelium of the newborn glomerulus to compensate for th© pauc ity of fenestrae through which glomerular filtrate can pass. The glomerular epithelial cells showed much reaction precip itate associated with the plasma membranes which were in apposition with other membranes, thus probably delineating the pathway for glomerular filtrate from the capillaries to Bowman's space. As with the adult, it is postulated that there are 42. probably at least two types of enzymes present in the new born kidney with pH optima at 7.2 and 9.4. At birth, the ATP ase active at pH 7.2 is probably abundant and stable and there fore more resistant to fixation effects. At pH 9.4, the effects of fixation on enzymatic activity of the newborn kidney were very apparent. We can only speculate as to why this is so. This enzyme with a pH optimum at 9.4 could be only present in small amounts, or was unstable, or was in an inactivated form, or a combination of all these three possibilities. With a diminished total plasma membrane surface area, it is conceivable to have a diminished amount of enzymes associated with it. During de velopment enzymes may change from the inactive form to the active form as well as change in stability (Moog, 1965). The increase in the quantity of enzymes demonstrable histochemic-ally could result from protein synthesis (Priestly and Malt, 1968) or an activation of enzymes previously in an inactive form. c- SUMMARY OF ENZYMATIC AVTIVITY OF THE ADULT AND NEWBORN  KIDNEY The functional capacity of both the adult and new born kidney can be correlated with the extent of its morpho logical differentiation and with the amounts of enzymes present. The adult kidney maintains body homeostasis efficient ly. The tubular elements in the cortex and the glomeruli are ultrastructurally complex. The amount of ATPase enzymatic activity demonstrable histochemically is considerable. The enzymes appear to be highly stable and are not affected by long periods of pre-fixation in glutaraldehyde prior to incubation in the ATP substrate medium. In the proximal tubules, the reaction precipitate is localised on the memb ranes of the brush border microvilli, t£e limiting membranes of some of the apical vesicles and vacuoles, and the membranes of the basal interdigitations. The brush border ATPase is probably related to the active transport across plasma membranes of small molecules from the tubular lumens into the 43. cells and vice versa (Loewy and Siekevitz, 1963). Larger mol ecules, colloidal materials and proteins perhaps enter the cells by pinocytosis. Some of the tubular invaginations from the bases of the microvilli and some of the apical vesicles and vacuoles sometimes show enzymatic activity. These cellular components associated with pinocytosis might represent a form of membrane flow within the cell which is energy-requiring (Loewy and Siekevitz, 1963). The basal infoldings divide the cytoplasm into numerous compartments within which are contained large, elongated mitochondria. These basal interdigitations are highly sensitive to conditions of fixation and to solut ions injected intravenously (Caulfield and Trump, 1962). It is suggested that the ATPases in the basilar parts of the proximal tubule cells are associated with the transport of salts and water. In the distal tubules, it is difficult to correlate the ATPase enzymatic activity present on the basilar infolding membranes with some of the postulated functions of the distal tubules, for example,.transport of sodium, secret ion of potassium and ammonia, and acidification of the urine (Ericsson and Trump, 1969). In the glomerulus, reaction precipitate is observed on the podocytic foot processes. Besides having a mechanical supportive function, t^re podocytic cells probably actively resorb proteins that have filtered through the endothelial fenestrae and basement membrane (Jones, 1969). The newborn kidney, as compared with the adult kidney, is functionally immature, morphologically undifferent iated and enzymatically less adequately endowed (Wacker et al., 1961; Pinkstaff et al., 1962; Moog, 1965; Wachstein and Brad shaw, 1965). Enzymatic activity that is demonstrable histo-chemically is localised on the lateral membranes between individual cells of the undifferentiated tubules, on the apposed membranes of podocytic cells and sometimes in the endothelium. The presence of these enzymes would show that the newborn kidney, though still immature, is capable of carrying out many of the functions essential for maintaining homeo-44. stasis. In the undifferentiated tubules there is no brush border with its complement of enzymes. This would perhaps ex plain, partially, the low rate of glucose absorption in the newborn kidney (Wachstein and Bradshaw, 1965). Newborn rats cannot eliminate excess water from their bodies as efficient ly as the adults (Pinkstaff et al., 1962; Wachstein and Brad shaw, 1965). If water is mainly transported across the memb ranes of the basilar interdigitations of the proximal tubules cells, then the lack or paucity of basilar infoldings with the corresponding absence of enzymatic activity, would account for the decreased functional capacity in terms of water movement. For both the adult and the newborn kidneys, it is postulated that there are at least two types of ATPases -localised at the plasma membranes with pH optima of 7.2 and 9.4. In the adult, the enzymes appear to be unaffected by pre-fixation in glutaraldehyde. In the newborn however, the enzyme active at pH 9.4 seems to be sensitive to the length of pre-fixation. D. NUCLEAR STAINING The significance of nuclear staining in tissues incubated for the ATPase enzymatic reaction is still in abey ance. Nuclear staining is frequently observed (Padykula and Herman, 1955a; Novikoff et al., 1958; Holt, 1959; Pinkstaff et al., 1962; Sandler and Bourne, 1962; Wachstein and Brad shaw, 1962; Ashworth et al., 1963; Deane, 1963; Tewari and Bourne, 1963a, 1963b; McClurkin, 1964; Wachstein and Besen, 1964; Klein, 1966; Moses et al., 1966; Tasuzumi and Tsubo, 1966; Jacobsen and Jorgensen, 1969). In most instances, as with the author's observations, nuclear staining is erratic and inconsistent and is considered as artifactual rather than as a manifestation of enzymatic activity. The mechanism of nuclear staining is not clear but the most commonly given reason is an affinity of nuclei for lead and calcium phosph ates (Ashworth et al., 1963; Deane, 1963; Moses et al., 1966). In some instances, investigators have found a consistent 45. pattern of nuclear staining. Pinkstaff et al. (1962) observed nuclear staining in the proximal tubules in all fetal and post-natal stages, and an increasing nuclear staining in dist al tubules. Sandler and Bourne (1962) could produce or cause to vanish nuclear staining by varying the magnesium sulfate concentration in the incubating media. Others have found nuc lear staining to be dependent on the concentration of ATP and of lead in the medium (Padykula and Herman, 1955a; Novikoff et al., 1958; Moses et al., 1966). Yasuzumi and Tsubo (1966) by modifying the histochemical method used cou-ld localise react ion precipitate in the region of the nuclear pores. Some spec ulations as to the function of this nuclear ATPase have been put forward. This ATPase, if present, would hint at a meta bolic interaction between the nuclei and the cytoplasm (Sand ler and Bourne, 1962; Tewari and Bourne, 1963a, 1963b; McClurkin, 1964; Yasuzumi and Tsubo, 1966). E. CONTROLS In practically all control specimens, no precipitate was observed. But occasionally there was precipitate assoc iated with the brush border of the proximal tubules (Persijn et al., 1961; Wachstein and Besen, 1964) when the method of Wachstein and Meisel (1957) was used. The precipitate is elec tron-dense but the nature of the composition of the precipit ate is not at all clear. In the present study, it was observed that there was a tendency for accumulation of precipitate to occur as the microvilli developed in length and in complexity. Perhaps with differentiation, the microvilli membranes acquire the glycocalyx coat which has an affinity for the precipitates formed as a result of the interaction between the various components of the incubating media. There does not appear to be this selective affinity for precipitates on the glomerular podocytic cell membranes where a coat of mucosubstances is also present (Jones, 1969). 46. ILLUSTRATIOHS Figures 1-4 Enzymatic activity of the adult kidney at pH 7.2 5 - 10 Enzymatic activity of the newborn kidney at pH 7.2 11 - 17 Enzymatic activity of the newborn kidney at pH 9.4 18-25 Effect of modifiers on enzymatic activity of the adult kidney 26 - 36 Effect of modifiers on enzymatic activity of the newborn kidney 37 _ 40 Controls with the Wachstein-Meisel method at pH 7.2- adult kidney 41 - 44 Controls with the Wachstein-Meisel method at pH 7.2 - newborn kidney ABBREVIATIONS L lumen C capillary N nucleus M mitochondrion E parietal epithelium Et erythrocyte p podocyte f podocytic foot process a small apical vesicle v large apical vacuole t tubular invagination m microvillus b brush border e endothelium bm basement membrane 47. 7 >V.> _ ' V- , _ ^ ; van aaa* , • . * • % « -—a M Figure 1 5 hours pre-fixation in glutaraldehyde. A proximal tubule. The brush border (b) of the proximal tubule from an adult kidney is exten sive. In response to the mode of fixation, the microvilli are closely packed together and the tubular lumen is obliterated. In the apical cytoplasm are a number of tubular invaginations (t), small apical vesicles (a) and larger apical vacuoles (v). The cytoplasmic compart ments, with associated mitochondria (M), form ed by the infoldings of the basal membranes extend almost up to the base of the brush bord er. Reaction precipitate is present in the brush border, in the cytoplasm and on the cytoplasmic aspect of the infolding membranes. x 11,800 48 Figure 2 5 hours pre-fixation in glutaraldehyde. A proximal tubule. Some of the interdigitat ions of the basal membranes are clearly shown. The extracellular compartments (arrows) formed by the infolding membranes are slightly en larged in response to the effects of fixation. The reaction precipitate, in the form of clumps, is found adhering to the interdigitating memb ranes and is also present in the basement membrane (bm). x 34,300 49. Figure 3 5 hours pre-fixation in glutaraldehyde. A distal tubule. There is a much more el aborate system of interdigitations of the basal membranes.A large number of elongated mitochondria (M) are closely associated with the membranes. The reaction precipitate is present on the cytoplasmic aspects of the membranes as well as within the cytoplasm. x 16,390 50. Figure 4 5 hours pre-fixation in glutaraldehyde. A glomerulus. The basement membrane (bm) separating the fenestrated endothelium (e) from the intricate pattern of podocytic foot processes (f) is prominent. One podocytic cell (p) may send out cytoplasmic processes to more than one capillary (arrows).Fine reaction pre cipitate is seen on the membranes and in the cytoplasm of the podocytic foot processes, in the basement membrane but not in the endothelium. x 11,800 51 Figure 5 2 hour-old kidney. 2 hours pre-fixation in glutaraldehyde. An undifferentiated tubule. There is intense enzymatic reaction on the lateral membranes between the individual cells. The plasma memb rane lining the luiften (L) shows no sign of enzymatic activity. Only one of the tubular cells (arrow) has reaction precipitate assoc iated with the simple basal membrane. x 4,700 52. Figure 6 2 hour-old kidney. 2 hours pre-fixation in glut arald ehyd e. An undifferentiated tubule. Reaction precipi tate is abundant on the lateral membranes. At the luminal surface (L) a small amount of precipitate is present on the plasma membrane of the few short microvilli (m) as well as witfrin the core of the microvilli. There is no reaction on the basal membranes. x 9,100 53. Figure 7 2 hour-old kidney. 2 hours pre-fixation in glut arald ehyd e. An undifferentiated tubule. The deposition of the reaction product for ATPase activity on the lateral membranes (arrows) clearly demar cates the lateral boundaries of each cell in the tubule. The basal membranes show some enzymatic activity. However, there is no pre cipitate on the luminal (L) surface plasma membrane. x 9,100 54. Figure 8 24 hour-old kidney. 5 hours pre-fixation in glutaraldehyde. A developing proximal tubule. Unlike the un differentiated tubule, the brush border (b) is relatively well-developed and there are a large number of small apical vesicles (a), large apical vacuoles (v) and mitochondria (M). In the lumen (L) there is a probably degenerating cell. The fine reaction precipitate encrusts the micro villi of the brush border (b). There is some reaction product on the membranes of the dev eloping basal interdigitations (arrows). x 4,700 55. Figure 9 2 hour-old kidney. 2 hours pre-fixation in glut arald ehyd e. An immature glomerulus. The podocytic cells are closely packed together. Reaction is most prominently observed where two sets of memb ranes are in apposition (arrows). x 9,100 56. Figure 10 24 hour-old kidney. 5 nours pre-fixation in glut araId ehyd e. A slightly more differentiated glomerulus than in Figure 9. The podocytic cell body (p) is still situated close to the capillary (C). There are a number of podocytic foot processes (f) abutting on the trilaminar basement membrane (bm). The endothelial cell (e) is becoming fenestrated (arrows). The reaction precipitate is found not only on the cell membranes but also within the cytoplasm of the podocytic cell body, the podocytic foot processes and endothelium. There is also quite a bit of precipitate assoc iated with the denser heterochromatin regions of the podocytic cell nucleus (N). x 11,800 57. Figure 11 2 hour-old kidney. 2 hours pre-fixation in glut arald ehyd e. An undifferentiated tubule. Morphologically the tubular cells are well preserved except for a few "exploded" mitochondria (M). With 2 hours pre-fixation, there is almost always no demonstrable enzymatic reaction. Occasion ally a few tubular cells show discrete part icles of reaction precipitate on the plasma membrane lining the lumen (L). There is no reaction on the lateral membranes (arrows) or basal membranes. x 9,100 58. Figure 12 1 hour-old kidney. 1 hour pre-fixation in glut arald ehyd e. An undifferentiated tubule. As in Figure 11 there is only a small amount of precipitate associated with the membrane lining the tub ular lumen (L). There is no enzymatic activity on the lateral membranes (arrows) or basal membranes. x 11,800 59. Figure 13 1 hour-old kidney. 15 mins. pre-fixation in glutaraldehyde. A developing proximal tubule. The microvilli (m), though still few in number, are becoming long and slender. Ultrastructurally the cells do not appear to be so well preserved as in Figures 11-12. However, with a shortened pre-fixation period in glutaraldehyde (15 mins. instead of 1 hour) enzymatic activity is pre sent on the microvilli membranes (m) and on the lateral membranes (arrows). There is some precipitate in the region of the basement membrane (bm). x 7,500 60. Figure 14 12 hour-old kidney. 2 hours pre-fixation in glut araId ehyd e. A developing proximal tubule. The microvilli (m) of the relatively well-developed brush border are coated with reaction precipitate deposits. The tubular invaginations (t) from the bases of the microvilli (m), the small apical vesicles (a) and some of the large ap ical vacuoles (v) also show the presence of reaction product. There is an intense reaction on the lateral membranes (arrows). At the base of the tubular cells, some precipitate is pre sent but it is not clear whether the precipi tate is associated with the basal membranes or the basement membrane. x 9,100 61. Figure 15 3 day-old kidney. 5 hours pre-fixation in glut arald ehyd e. Brush border of a developing proximal tubule. The brush border (b) is quite extensive. It is in a collapsed state as a result of the imm ersion method of fixation used. It is clearly seen that the fine precipitate encrusts the membranes of the microvilli. A number of the tubular invaginations from the bases of the micro villi (arrows) show a marked deposition of the reaction precipitate. The few small apical vac uoles (v) that are present have no reaction precipitate. x 11,800 Figure 16 12 hour-old kidney. 2 hours pre-fixation in glut araId ehyd e. An immature glomerulus. This group of six podocytic cells show variations in response to incubation in the ATP substrate medium. Some cells show a weak reaction (l, 2) while others a more pronounced reaction (3, 4, 5). The podo-cyte in the centre (6) is not reactive except where its cell membrane is in apposition with the cell membrane of another podocyte (5) (arrow). x 7,500 63. Figure 17 12 hour-old kidney. 2 hours pre-fixation in glut araId ehyd e. An immature glomerulus. The podocytic cells (p) are closely packed together at the basal poles but the apical poles are free in Bowman's space (L). There are no foot processes. The endothelium (e) has not assumed its fenestrated form. It is very apparent that there is an intense ATPase reaction where the membranes of the podocytic cells are in apposition with the membranes of the neighbouring cells (arrows), but not if they are free and exposed in Bow man' s space (L). The endothelial cells (e) have no reaction precipitate. x 4,700 64. Figure 18 3 hours pre-fixation in glutaraldehyde. Pre incubation exposure to PHMB. Incubation with PHMB at pH 7.2. Two proximal tubules. Though adjacent to one another, the two tubules show variations in enzymatic activity. In one tubule there are two distinct regions of enzymatic activity; at the brush border (b) and basal interdigit ations (arrows). The basal infoldings of the other tubule show no deposition of reaction precipitate. x 7,500 65. Figure 19 3 hours pre-fixation in glutaraldehyde. No pre incubation exposure to PHMB. Incubation with PHMB at pH 7.2. Brush border of a proximal tubule. There is some indication of an electron-dense material within the core of the microvilli (circle). The reaction precipitate is not uniformly dist ributed over the membranes of all the microvilli in the brush border. Instead clumps of precipit ate are observed in various regions of the brush border (arrows). This is probably artifactual rather than a result of heterogeneity of response to PHMB. x 11,800 66. Figure 20 3 hours pre-fixation in glutaraldenyde. No pre incubation exposure to PHMB. Incubation with Pfflffl at pH 7.2. Glomerulus. This is a low magnification electron micrograph of a glomerulus showing various cap illaries (C) and the intricate network of podo cytic foot processes (f) on the trilaminar base ment membrane separating the endothelium (e) from the visceral epithelium. The reaction pre cipitate is distributed all along the plasma membranes of the podocytic foot processes but not on the membranes of the endothelial cells. x 3,700 67. Figure 21 3 hours pre-fixation in glutaraldehyde. Pre incubation exposure to PHMB. Incubation with PHMB at pH 7.2. Glomerulus. The fenestrated endothelium (arrows) of the capillaries (C) is separated from the podocytic foot processes (f) by a distinctly visible basement membrane (bm). There is no reaction precipitate on the endothelium. How ever reaction precipitate is present all along the plasma membranes of the podocytic foot processes. x 9,100 68. Figure ZZ 3 hours pre-fixation in glutaraldehyde. Pre incubation exposure to PHMB. Incubation with PHMB at pH 9.4. Proximal tubule. The basal interdigitations in the proximal tubule are quite extensive. Some may be confined to the basal portions of the cell while others may extend almost to the bases of the brush border (b). The extracellular compart ments formed by the interdigitations are very sensitive to the effects of fixation and are often separated, as exemplified here. There is no ATP ase activity on these membranes although enzy matic activity is readily observed in the brush border (b) and in the tubular invaginations (arrows) arising from the bases of the microvilli. Only one apical vacuole (v) has some reaction precipitate associated with it. x 5,700 69. Figure 25 3 hours pre-fixation in glutaraldehyde. No pre incubation exposure to PHMB. Incubation with PHMB at pH 9.4. Proximal tubule. The enlarged extracellular compartments (arrows) bound by the infolding basal membranes indicate the sensitivity of the basal regions of the proximal tubules to the effects of fixation. Not all the extracellular compartments are enlarged to the same extent. Unlike Figure 22, the basal interdigitations show an intense enzymatic reaction. The fine reaction precipitate is seen adhering to the membranes and is not free in the extracellular space. The basement membrane (bm) is relatively thick and is generally free of any precipitate. x 11,800 70. Figure 24 3 hours pre-fixation in glutaraldehyde. Pre incubation exposure to PHMB. Incubation with PHMB at pH 9.4. Brush border of a proximal tubule. There is an abundant deposition of reaction precipitate along the membranes of the microvilli making up the brush border. A large number of the tubular invaginations from the bases of the microvilli (arrows) are also coated with the reaction pre cipitate. Some small apical vesicles (a) have reaction product on their limiting membranes. The large apical vacuoles (v) that are present have no accumulation of reaction precipitate. x 11,800 Figure 25 3 hours pre-fixation in glutaraldehyde. No pre incubation exposure to PHMB. Incubation with PHMB at pH 9.4. Glomerulus. The fine reaction precipitate is observed not only along the membranes of the podocytic foot processes (f) but also within the cytoplasm (compare with Figures 20, 21). There is some precipitate in the basement memb rane (bm) which is probably due to diffusion of reaction precipitate from the adjacent podo cytic foot processes. The fenestrated endo thelium (e) shows no enzymatic activity. x 16,300 Figure 26 2 hour-old kidney. 2 hours pre-fixation in gluta raldehyde. No pre-incubation exposure to PHMB. Incubation with PHMB at pH 7.2. An undifferentiated tubule. The plasma membrane lining the lumen (L) as well as the basal memb ranes (arrows) of the tubular cells are still simple in contour. The lateral membranes show the beginnings of interdigitations which will become much more complex with continuing diff erentiation. Heavy deposits of reaction pre cipitate on the lateral membranes accentuate the lateral boundaries between the tubular cells There is no reaction precipitate on the basal membranes or on the luminal surface membrane. x 4,700 73. Figure 27 2 hour-old kidney. 2 hours pre-fixation in gluta raldehyde. Pre-incubation exposure to PHMB. Incubation with PHMB at pH 7.2. An undifferentiated tubule. The outline of the luminal (L) surface membrane is still relatively smooth although a few microvilli are present. The basal membranes of some cells are beginning to infold (arrows). The lateral membranes also show some interdigitations. There is intense enzymatic reaction all along the luminal surface plasma membrane, the interdigitating lateral membranes and the membranes of the basal infold-ings, wherever they are present. A few of the nuclei (N) show an accumulation of fine precipitate. x 5,700 74. Figure 28 2 hour-old kidney. 2 hours pre-fixation in gluta raldehyde. Pre-incubation exposure to PHMB. Incubation with PHMB at pH 7.2. An undifferentiated tubule. Ultrastructurally the tubular cells are simple. Each cell consists of a large nucleus (N) and a few mitochondria (M) contained within a small amount of cytoplasm. At the at»ex of the cell there are a few micro villi (m). Part of the apical cytoplasm appears to be blebbing off (arrows) and discarded into the lumen (L) as cellular debris. The lateral membranes follow a straight path from the lumen to the base of the cell. The basal membranes show no complex interdigitations as in the adult. There is practically no demonstrable enzymatic activity except for part of the lateral membrane, (circle). x 7,500 75. Figure 29 2 hour-old kidney. 2 hours pre-fixation in gluta raldehyde. No pre-incubation exposure to PHMB. Incubation with PHMB at pH 7.2. An undifferentiated tubule. All, but one, of the cells is normal and show deposition of re action precipitate on the lateral membranes. This one cell is "exploded." The contents of the cell, that is, the cytoplasm and mitochondria (M) are being spilled out into the lumen (L). The nucleus (N) is swollen to immense proportions. The nuc lear membrane (arrows) appears intact. The chrom atin material adheres to the nuclear membrane or is suspended in the nucleoplasm. The reason for this particular cell1s extreme sensitivity to experimental conditions is not known. x 7,500 76. •Figure 50 2 hour-old kidney. 2 hours pre-fixation in gluta raldehyde. No pre-incubation exposure to PHMB. Incubation with PHMB at pH 7.2. A developing proximal tubule. The tubule shows the first definite signs of differentiation into a proximal tubule. The microvilli (m) are still sparse but relatively long and slender. There are a number of apical vesicles (a) in the apical cytoplasm. The lateral and basal membranes are simple in contour. Reaction precipitate is deposited heavily on the lateral membranes (arrows), the luminal (L) surface membrane and the membranes of the microvilli. Some of the small apical vesicles (a) have reaction precipitate all along the limit ing membranes. x 7,500 77. Figure 51 2 hour-old kidney. 2 hours pre-fixation in gluta raldehyde. Pre-incubation exposure to PHI-IB. Incubation with PHMB at pH 7.2. A developing proximal tubule. This is a tubule further along in differentiation (compare with Figure 30). The brush border (b) is quite well-developed, the lateral membranes pursue a slightly more tortuous course from the lumens to the base of the cell (arrows) and some interdigitations of the basal membranes are seen (circle). There are a large number of tubular invaginations (t) from the bases of the microvilli, small apical vesicles (a) and large apical vacuoles (v). Some of the small, round mitochondria (M) that are present appear to be sensitive to the effects of fixation. Enzymatic activity is most prominent in the brush border (b), the tubular invaginations (t), and on the lateral and basal interdigitations. x 3,700 78. Figure 52 2 hour-old kidney. 2 hours pre-fixation in gluta raldehyde. Pre-incubation exposure to PHI-IB. Incubation with PHMB at pH 7.2. A developing proximal tubule. This tubule is more differentiated than those in Figures 30 and 51. The brush border (b) and the basal inter digitations are quite extensive. Cytoplasmic compartments with associated mitochondria (M) are developing. Most of the mitochondria are still small and round (Ml, M2) while others are becoming elongate (M5, M4). The reaction precipitate is observed on the membranes of the basal interdigitations, the brush border, tub ular invaginations (t) and small apical vesicles Ca). x 9,100 79. e Figure 55 2 hour-old kidney. 2 hours pre-fixation in gluta raldehyde. Fo pre-incubation exposure to PHMB. Incubation with PHMB at pH 7.2. An immature glomerulus. The parietal epithelium (E-)- delineates the outermost extent of the glom erulus. The visceral epithelium, made up of the podocytic cell bodies (p) and the beginnings of foot processes (f), surround the endothelium (e) of two capillaries. The capillary lumens are small but can be recognised by the presence of an erythrocyte (Et). The apical poles of the podo cytic cells are free in Bowman's space (L) while the foot processes from the basal poles abutt on the basement membrane around the capillaries. Enzymatic activity is only present on the memb ranes of the foot processes in direct contact with the basement membrane, and also on the lateral membranes of the parietal epithelial cells. x 3,700 80. Figure 54 2 hour-old kidney. 2 hours pre-fixation in gluta raldehyde. No pre-incubation exposure to L-cysteine. Incubation with L-cysteine at pH 7.2. An undifferentiated tubule. The tubule is in an early stage of development. The lumen (L) cont aining some cellular debris (arrow) is still small. A tubule consisting of a single layer of cells is probably formed from the growth and movement of such a group of undifferentiated cells. Then these undifferentiated cells acquire the ultra structural features characteristic of each portion of the nephron. The deposition of reaction preci pitate is abundant all along the lateral membranes separating each individual cell. There is also some precipitate on the luminal surface membranes. Only two of the nuclei (N) appear to have fine precipitate associated with them. x 5,700 81. Figure 55 2 hour-old kidney. 2 hours pre-fixation in gluta raldehyde. No pre-incubation exposure to L-cysteine. Incubation with L-cysteine at pH 7.2. An undifferentiated tubule. Occasionally a cilium (arrow) is seen arising from the apical plasma membrane. Otherwise the luminal plasma membrane is simple in contour. A piece of cytoplasmic material (X) appears to be extruded into the lumen (L). Only the lateral membranes between various cells show an abundant deposition of reaction precipitate. x 16,500 82. Figure 56 2 hour-old kidney. 2 hours pre-fixation in gluta raldehyde. No pre-incubation exposure to L-cysteine. Incubation with L-cysteine at pH 7.2. An immature glomerulus. The podocytic cells (p) are still closely packed together. The cyto plasm of some of the cells are beginning to dev elop into foot processes. There is intense enzy matic reaction on practically all the membranes whether they are in apposition or are free. Some of the free membranes show a lesser amount of or no precipitate (arrows). In one podocytic cell (pi) there is also some precipitate in the cytoplasm. x 7,500 83. Figure 57 Brush border of a proximal tubule.The brush border (b) is very well-developed. The micro villi are long and slender and are closely packed together. This collapsed condition of the brush border is artifactual and is a result of the mode of fixation used. The tubular in vaginations from the bases of the microvilli appear to contain a dense material (arrows). There are no apical vesicles or vacuoles in the apical cytoplasm of this particular cell. There is no accumulation of electron-dense precipitate except for a few specks here and there. x 9,100 84. Figure 58 Distal tubule. The basal membranes infold ex tensively dividing the cytoplasm into numerous compartments within which are contained elongated mitochondria (M). The extracellular compartments are enlarged slightly probably in response to fixation conditions (arrows). On the luminal surface of the cell there are a few short microvilli (m). In the apical cytoplasm there are a few small apical vesicles (a). There is no precipitate on the basal interdigitations, on the microvilli or within the cytoplasm. x 7,500 85. A glomerulus. The lumens of three capillaries (C) are seen in this electron micrograph. The endothelium (e) lining the capillaries is fen estrated. A thick basement membrane (bm) is interposed between the endothelium and the podo cytic foot processes (f), which are cytoplasmic prolongations of the podocytic cells (p). The endothelium, the basement membrane and the podo cytic foot processes are the three components of the filtration apparatus. No precipitate is observed within the glomerulus. x 9,100 86. Figure 40 A proximal tubule. The brush border (b) is well-developed. In the apical cytoplasm there are a large number of small apical vesicles (a) and large apical vacuoles (v). Numerous mitochondria (M) are present in the cytoplasmic compartments formed by the elaborate interdigitations of the basal membranes. The extracellular compartments are enlarged in response to the conditions of fixation. Some electron-dense precipitate is observed in the region of the brush border (compare with Figures 1 and IS). x 7,500 87 Figure 41 An undifferentiated tubule. The tubular cells are ultrastructurally simple. The nuclei (N") are large. In the cytoplasm there a few small round mitochondria (M). The luminal surface membrane is simple in contour. Part of the apical cytoplasm seems to oe blebbing off (arrow) and being discarded into the lumen (L) as debris. Eo precipitate is present on any of the plasma membranes. x 9,100 88. Figure 42 Brush "border of a developing proximal tubule. The brush border (b) is relatively well-developed. There are a large number of long slender microvilli. Tubular invaginations (t), small apical vesicles (a) and large apical vacuoles (v) are present in the apical cyto plasm. There are also a few mitochondria (M). There is no precipitate present. x 11,800 89. Figure 45 An immature glomerulus. The parietal epithelium (E) is the outermost component of the glomerulus. Within Bowman's space (L) are the podocytic cells (p). A large nucleus is present in the apical poles of the cells. Podocytic foot processes (f) arise from the basal portions of the cells and abutt onto the basement membrane separating the podocytes from the endothelium (e). The cap illary lumen (C) is still small. There is no deposition of precipitate in the glomerulus. (The dark chunks are dirt particles). x 4,700 90. Figure 44 A developing proximal tubule. The microvilli (m), though still few in number, are long and slender. Tubular invaginations (t), small apical vesicles (a) and large apical vacuoles (v) are present in the apical cytoplasm. There are quite a few mitochondria (M), but these appear to be particularly susceptible to the effects of fix ation. Electron-dense precipitate is seen adhering to the microvilli membranes and some of the tub ular invaginations and apical vesicles. The amount of precipitate is less than in tissues incubated in a medium containing ATP as a substrate. (Compare with Figures 8, 30-32). x 7,500 91. BIBLIOGRAPHY Abel, J.H.: Electron microscopic demonstration of adeno sine triphosphate phosphohydrolase activity in herring gull salt glands. J. Histochem. OytOchem. 17:570-584 (1969). Anderson, W.A.: Cytochemistry of sea urchin gametes I Intramitochondrial localisation of glycogen, glucose-6-pMsphatase and ATPase activity in spermatozoa of Paracentrotus lividus. J. Ultrastruct. Res. 24:398-411 (1968). 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