Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Histochemical localisation of adenosine triphosphatase activity in adult and newborn rat kidneys at the.. 1969

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
UBC_1970_A6_7 L55.pdf [ 17.91MB ]
UBC_1970_A6_7 L55.pdf
Metadata
JSON: 1.0101997.json
JSON-LD: 1.0101997+ld.json
RDF/XML (Pretty): 1.0101997.xml
RDF/JSON: 1.0101997+rdf.json
Turtle: 1.0101997+rdf-turtle.txt
N-Triples: 1.0101997+rdf-ntriples.txt
Citation
1.0101997.ris

Full Text

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 i n the Department of Anatomy We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1969 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study . I f u r t h e r agree tha permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department o r by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department of Anatomy The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date November, 1969 i . ABSTRACT The. histochemical l o c a l i s a t i o n of ATPase enzymatic a c t i v i t y at the l e v e l of the electron microscope was carried out on adult and newborn kidney tissue pre-fixed i n 55̂  g l u t a - 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 ef f e c t s of the modifiers PHMB and L-cysteine were also studied. In the adult rat kidney, the observations of other investigators on kidney ATPase a c t i v i t y were substantiated. Reaction p r e c i p i t a t e was l o c a l i s e d at the brush border of the proximal tubules, the membranes of the basal and l a t e r a l i n t e r d i g i t a t i o n s of the proximal and d i s t a l tubules, and the plasma membranes of the podocytic foot processes. PHMB exerted an i n h i b i t o r y effect on d i s t a l tubular a c t i v i t y at both pH 7.2 and pH 9.4, while cysteine was i n h i b i t o r y only at pH 9.4. Glomerular ATPase a c t i v i t y was in h i b i t e d by PHMB and L-cysteine at pH 9.4. In the newborn rat kidney, ATPase enzymatic act- ' i v i t y was observed i n the tubular elements as well as i n the glomeruli. In the undifferentiated tubules, reaction product was abundant on the l a t e r a l membranes between i n d i v - i d u a l c e l l s . The luminal and basal plasma membranes, which • were simple i n contour, showed l i t t l e or no accumulation of pr e c i p i t a t e . However, as the m i c r o v i l l i became long and slender i n the early stages of the d i f f e r e n t i a t i o n of the brush border, there was a concomitant increase i n the intens- ity-oof the ATPase enzymatic reaction. Si m i l a r l y , reaction product became associated with the developing basal i n t e r - d i g i t a t i o n s . In the immature glomerulus, reaction p r e c i p i t - ate was most often observed where two sets of membranes were i n apposition. With d i f f e r e n t i a t i o n , enzymatic a c t i v i t y was l o c a l i s e d primarily on the podocytic foot processes. The l o c a l i s a t i o n of ATPase a c t i v i t y at pH 9.4 was found to be influenced by the time of pre- f i x a t i o n i n glutaraldehyde 0 while ATPase a c t i v i t y at pH 7.2 was not affected. At pH 7.2, neither tubular nor glomerular ATPase enzymatic a c t i v i t y responded to PHMB or L-cysteine. For both adult and newborn kidneys, the c o r r e l a t - ion between structure and function was b r i e f l y considered. The adult kidney i s an important and e f f i c i e n t homeostatic organ. In urine formation various substances are transport- ed across the c e l l membranes of the glomeruli and tubules. U l t r a s t r u c t u r a l l y , the glomeruli and tubules show modifi- cations c h a r a c t e r i s t i c of c e l l s engaged i n active trans- port processes. There i s a large increase i n plasma memb- rane surface area, as exemplified by the i n t r i c a t e i n t e r - d i g i t a t i o n s of the podocytic foot processes, the elaborate basal and l a t e r a l infoldings, and the brush border of the proximal tubules. Much ATPase a c t i v i t y was found associated- with these plasma membranes. The newborn kidney i s not as e f f i c i e n t as the adult kidney i n maintaining body homeo- s t a s i s . I t i s not only f u n c t i o n a l l y but also morphologically immature. Most of the tubules and glomeruli are undifferen- t i a t e d and do not show s p e c i a l i s a t i o n s of the plasma membranes as seen i n the adult kidney. There i s also a r e l a t i v e l y smaller amount of ATPase present i n the newborn kidney. For both adult and newborn kidneys, i t was postulated that at leas t two types of ATPases with d i f f e r e n t pH optima are present on the plasma membranes of the tubules and glomeruli. i i i . TABLE OF CONTENTS Page I Introduction 1 II Materials and methods 7 A. Materials 7 B. P r e - f i x a t i o n i n glutaraldehyde 7 C. Incubation f o r histochemistry 9 (a) Preparation of incubating medium at pH 7.2 9 (b) Preparation of incubating medium at pH 9.4 9 D. E f f e c t of modifiers 10 E. Controls 10 P. Po s t - f i x a t i o n i n osmium 11 G. Eleo'tron microscopy 11 I I I Observations 12 A. Morphological basis f o r histochemical interpretations 12 (a) Adult kidney 12 (b) Newborn kidney 15 B<. E f f e c t of f i x a t i o n on u l t r a structure 17 (4) Adult kidney 17 (b) Newborn kidney 18 C. Ef f e c t of f i x a t i o n on enzymatic a c t i v i t y 19 D. Enzymatic a c t i v i t y of the adult kidney 21 (a) pH 7.2 - lead method 21 (b) pH 9.4 - calcium method 23 E. Enzymatic a c t i v i t y of the newborn kidney 23 (a) pH 7.2 - lead method 23 (b) pH 9.4 - calcium method 25 F. E f f e c t of modifiers PHMB and L-cysteine on enzymatic a c t i v i t y 27 (a) Adult kidney 27 (b) Newborn kidney 29 i v . $ s Page G. Controls 30 H . Nuclear staining 30 IV Discussion 32 A. General 32 B!. Plasma membrane enzymatic a c t i v i t y 35 (a) Adult kidney 35 (b) Newborn kidney 40 C. Summary of enzymatic a c t i v i t y of the adult and newborn kidney 42 D. Nuclear staining 44 E. Controls 45 V I l l u s t r a t i o n s 46 VI Bibliography 91 V. TABLES Page I Age of the animals and the length of f i x a t i o n of the respective kidneys. 8 II The effect of f i x a t i o n on enzymatic a c t i v i t y . 20 III The effect of the modifiers, PHMB and L- cysteine, on enzymatic a c t i v i t y of the adult kidney. 27 IV The eff e c t of the modifiers, PHMB and L- cysteine, on enzymatic a c t i v i t y of the newborn kidney. 29 v i . ILLUSTRATIONS Figures Pages 1 - 4 Enzymatic a c t i v i t y of the adult kidney at pH 7.2. 47 - 50 5 - 10 Enzymatic a c t i v i t y of the newborn kidney at pH 7.2. 51 - 56 11 - 17 Enzymatic a c t i v i t y of the newborn kidney at pH 9.4. 57 - 63 18 - 25 Ef f e c t of modifiers on enzymatic a c t i v i t y of the adult kidney. 6 4 - 7 1 26 - 36 Ef f e c t of modifiers on enzymatic a c t i v i t y of the newborn kidney. 7 2 - 8 2 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 v i i . ACKNOWLEDGMENT I would l i k e to express my appreciation to my advisor, Dr. William A. Webber, f o r h i s guidance through- out the course of t h i s investigation, and f o r suggestions and constructive c r i t i c i s m s i n the preparation of the thesis; and to Mrs. Janice Blackbourn, f o r h e l p f u l hints i n the techniques of electron microscopy. 1. I INTRODUCTION The l o c a l i s a t i o n of enzymatic a c t i v i t y using "bio- chemical methods on homogenised tissu e f r a c t i o n s i s a means of assaying q u a n t i t a t i v e l y the various properties of enzymes under conditions approaching t h e i r n a t ural state. These f r a c t - ionation studies, though u s e f u l and indispensable, have an inherent l i m i t a t i o n . They show the association of enzymes with c e r t a i n types of c e l l u l a r components, but give no i n d i c - ation of the act u a l d i s t r i b u t i o n of the enzymes within i n d i - v i d u a l c e l l s or within groups of c e l l s . At the present time, a histochemical or cytochemical technique f o r emzyme l o c a l - i s a t i o n represents the only way of determining the s i t e s of enzymatic a c t i v i t y under i n s i t u or near i n s i t u conditions (Marches!, 1968). The main advantage of using a histochemical technique f o r enzyme l o c a l i s a t i o n i s that c e l l u l a r r e l a t i o n - ships are maintained i n an intact tissue section. It i s there- fore possible to v i s u a l i s e the products of an enzymatic react- ion i n r e l a t i o n to s p e c i f i c c e l l u l a r components, f o r example, mitochondria, n u c l e i or membranes. This has only been made fe a s i b l e by vast improvements i n the f i e l d of electron micro- scopy. Histochemistry at the l e v e l of the electron microscope, opens up Large areas i n c e l l u l t r a s t r u c t u r a l research. We must however, bear i n mind the l i m i t a t i o n s of existing* methods f o r the u l t r a s t r u c t u r a l l o c a l i s a t i o n of enzymatic a c t i v i t y . There needs to be adequate preservation of both the u l t r a - structure and the enzymatic a c t i v i t i e s of c e l l s and t i s s u e s . Ideally, fresh t i s s u e should be used, but t h i s i s not possible owing to extremely poor preservation of c e l l u l t r a s t r u c t u r e . A compromise i s made by a b r i e f p r e - fixation i n formaldehyde (Kaplan and Novikoff, 1959; Holt and Hicks, 1961), or more commonly, i n glutaraldehyde (Sabatini et a l . , 1963; 1964), p r i o r to incubation. The effect of f i x a t i o n on the character- i s t i c s of enzymes, whether quantitative or q u a l i t a t i v e or 2. both., i s not known at the present time. Besides f i x a t i o n , the tissue undergoes rigorous conditions of dehydration, i n f i l t r - ation, embedding and polymerisation i n an oven, i n preparat- ion f o r i t s examination under the electron beam (Sjostrand, 1967). How these processes a f f e c t the tissue are poorly, i f at a l l , understood. Nevertheless, i f these l i m i t a t i o n s are borne i n mind, much information can be gleaned from electron microscopical histochemical data. Glenner (1968) defines the histochemical system as "that system i n which intact tissue sections are incubated i n a solution containing a substrate, and the enzyme-catalised formation of the f i n a l reaction product (a precipitate) can be demonstrated i n s i t u by means of a va r i e t y of techniques including l i g h t , fluorescent, electron and interference micro- scopy." Many histochemical methods, to date, are based on simple metal-salt p r e c i p i t a t i o n reactions. The two common pro- cedures f o r the histochemical l o c a l i s a t i o n of adenosine t r i - phosphatase (ATPase) i n tissue sections use lead ions (Wachs- t e i n and Meisel, 1957) or calcium ions (Padykula and Herman, 1955a, 1955b; Padykula and Gauthier, 1965) to form a p r e c i p i - tate with the inorganic phosphate which i s released by the enzymatic hydrolysis of the substrate i n the incubation media, that i s , adenosine triphosphate (ATP). The reaction product, which i s electron dense and can be viewed d i r e c t l y under the electron microscope, i s presumably deposited at, or very near to, the act u a l s i t e of the enzymatic a c t i v i t y . This issue of whether the s i t e of deposition of the reaction p r e c i p i t a t e i s the actual s i t e of enzymatic a c t i v i t y , has not been solved yet, as many investigators stress (Otero-Vilardebo et a l . , 1963; Wachstein and Besen, 1964; Mao and Nakao, 1966). Most c e l l s store energy i n the form of high energy phosphate bonds i n ATP (Stumpf, 1953). This energy can be re- leased f o r use i n performing various c e l l u l a r a c t i v i t i e s by the enzyme ATPase. Therefore, the l o c a l i s a t i o n of the enzyme u l t r a s t r u c t u r a l l y would perhaps lead to some understanding of the mechanisms underlying c e l l u l a r function. Since the introduction of the lead (Wachstein and Meisel, 1957) and calcium (Padykula and Herman, 1955a, 1955b; Padykula and Gauthier, 1963) methods f o r the histochemical l o c a l i s a t i o n of ATPase, many s i t e s i n various tissues have been demonstrated to possess t h i s enzyme. This has been shown both with l i g h t microscopy (Padykula and Herman, 1955a, 1955h; Wachstein, 1955; Wachstein and Meisel, 1957; Wachstein et a l . , I960, 1962; Padykula and Gauthier, 1963; Tewari and Bourne, 1963a, 1963b; McClurkin, 1964; Wachstein and Besen, 1964; Far- quhar and Palade, 1966; Moses et a l . , 1966; Gauthier, 1967; Jacobsen et a l . , 1967; Brooke and Kaiser, 1969) and electron microscopy (Essner et a l . , 1958; Kaplan and Novikoff, 1959; P e r s i j n et a l . , 1961; Lazarus and Barden, 1962; Ashworth et a l . , 1963; Oterb-Vilardebo et a l . , 1963; Torack'and Barrnett, 1963; Wachstein and Besen, 1963; Goldfischer et a l . , 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 l o c a l i s a t i o n of t h e i r ATPase content and speculations as to the possible or probable roles i n the func- t i o n of t h e i r respective tissues have sometimes been put f o r - ward. A few examples from t h i s vast area of research are pre- sented. The l i t e r a t u r e cited i s by no means extensive nor complete. In s t r i a t e d muscles there i s a constant turnover of energy associated with the processes of contraction and r e - laxation. Many of these c e l l u l a r functions require the s p l i t t - ing of ATP with the concomitant release of energy. Following t h i s l i n e of thinking, i t i s expected that ATPase a c t i v i t y w i l l be demonstrable histochemically i n muscular ti s s u e . In f a c t , histochemical data on muscle ATPase i s available (Pady- kula and Herman, 1955a, 1955b; Padykula and Gauthier, 1963; Barden and Lazarus, 1964; Gauthier and Padykula, 1965; Kle i n , 1966; Gauthier, 1967; Grossman and Heitkamp, 1968; Brooke and Kaiser, 1969; Ogawa and Mayahara, 1969). As many as four d i f f e r e n t ATPases have been l o c a l i s e d i n s k e l e t a l muscle f i b r e s ; a mitochondrial and a m y o f i b r i l l a r ATPase, and two sarcotubular ATPases (Padykula and Gauthier, 1963; Gauthier and Padykula, 1965; Gauthier, 1967)..It was possible to d i f f e r e n - t i a t e among these four enzymes i n terms of t h e i r pH optima and t h e i r response to activators and i n h i b i t o r s . Of p a r t i c u l a r i n - terest i n the muscle are the two s i t e s of sarcotubular a c t i v i t y , one at the region of the t r i a d and the other at the region of the H band (Gauthier and Padykula, 1965; Gauthier, 1967). It i s suggested that the ATPase at the t r i a d may be involved i n the accumulation of calcium ions during the period of relaxa- t i o n , as well as i n the release of calcium ions following a stimulus f o r muscular contraction. The ATPase at the H band i s more s p e c i f i c a l l y associated with the rebinding of calcium ions during relaxation (Gauthier and Padykula, 1965; Gauthier, 1967). The l i v e r i s an important and a f u n c t i o n a l l y active organ whose diverse a c t i v i t i e s contribute to the s t r u c t u r a l and f u n c t i o n a l s t a b i l i t y of the whole organism. Morphologically i t i s a simple organ but metabolically i t i s not so. Many i n - vestigators have applied the histochemical methods f o r the l o c - a l i s a t i o n of ATPase to the l i v e r (Padykula and Herman, 1955a, 1955b; Essner et a l . , 1958; Novikoff et a l . , 1958; Holt and Hicks, 1961; Novikoff et a l . , 1961; P e r s i j n et a l . , 1961; .Wa-ah- stain and Bradshaw, 1962; Ashworth et a l . , 1963; Wachstein and Fernandez, 1964; Moses et a l . , 1966; Will s , 1967). The products of the enzymatic reaction have been found i n the membranes of the endoplasmic reticulum (Wachstein and Fernandez, 1964) but more commonly on the membranes of the m i c r o v i l l i of the b i l e c a n a l i c u l i (Essner et a l . , 1958). The possible p a r t i c i p a t i o n of these ATPases i n molecular transport and/or pinocytosis i s suggested (Essner et a l . , 1958). From biochemical studies i t was found that nervous tissues possess a large amount of the enzyme ATPase (Bonting et a l . , 1962). However, biochemical data i s only s t a t i s t i c a l and does not indicate the s i t e s of enzymatic a c t i v i t y . Histo- chemical methods are therefore employed to l o c a l i s e the ATPase a c t i v i t y i n various components of the nervous system (Novikoff et a l . , 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 n e u r o g l i a l dendrites ad- jacent to the c e l l body, synaptic terminals and g l i a l foot pro- cesses adjacent to c a p i l l a r y walls. They put forward the idea that the ATPase present on the membranes of the g l i a l processes may be related to the supposed function of the astrocytes i n transporting materials between blood vessels and neurons. At the synapses the enzyme may p a r t i c i p a t e i n the synthesis of acetylcholine. Interestingly enough, there i s no enzymatic act- i v i t y i n the endothelial c e l l s of the cerebral c a p i l l a r i e s , that i s , those of the blood-brain b a r r i e r , while enzymatic act- i v i t y i s present i n other endothelia. Involvement i n tr a n s f e r mechanisms across the walls of small blood vessels i s implied. Water, s a l t s and nutrients cross the c a p i l l a r y endothelium to the surrounding c e l l s by active transport and pinocytosis. Simultaneously, waste products from the c e l l s are removed i n the blood by s i m i l a r processes. Probably the enzyme ATPase i s necessary f o r these processes to occur. Perhaps one of the causes f o r the f a i l u r e of an interchange of materials between the en- d o t h e l i a l c e l l s of the cerebral c a p i l l a r i e s and the brain c e l l s , - i s the lack of the enzyme ATPase leading to an i n a b i l i t y of the endothelial c e l l s to carry out transport processes. ATPase a c t i v i t y has been l o c a l i s e d histochemically i n p r a c t i c a l l y every system i n the vertebrate body (Novikoff et a l . , 1961; Wachstein and Bradshaw, 1962; Ashworth et a l . , 1963; Barden and Lazarus, 1963; Bradshaw et a l . , 1963; Otero- Vilardebo et a l . , 1963; Goldfischer et a l . , 1964; Lazarus and Barden, 1964; Wachstein and Fernandez, 1964; Wheeler and Whi- ttam, 1964; Essner et a l . , 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 p a r t i c u l a r emphasis om' the kidney, i s no exception (Padykula and Herman, 1955b; Spater et a l . , 1958; Freiman and Kaplan, 1959; Kaplan and Novikoff, 1959; Freiman and Kaplan, I960; Novikoff et a l . , 1961; Pe r s i j n et a l . , 1961; Pinkstaff et a l . , 1962; Wachstein and Bradshaw, 1962; Ashworth et a l . , 1963; Otero-Vilardebo et a l . , 1963; Wachstein and Besen, 1963, 1964; Wheeler and Whittam, 1964; Wachstein and Bradshaw, 1965; Jacobsen et a l . , 1967; Abel, 1969; Jacobsen and Jorgensen, 1969). Most of the research on kidney ATPase, both with the l i g h t microscope and the electron microscope, has been on adult kidneys. Few studies have been carried out on newborn or developing kidneys (Pinkstaff et a l . , 1962; Wachstein and Bradshaw, 1965). These have only been at the l e v e l of the l i g h t microscope. Up to date, there i s no description of ATPase act- i v i t y i n newborn kidneys at the l e v e l of the electron micros- cope, to the knowledge of the author. The present study i s an attempt to confirm the ob- servations of other investigators on the l o c a l i s a t i o n of the re- action product f o r ATPase i n adult kidneys using both the lead (Wachstein and Meisel, 1957) and calcium (Padykula and Herman, 1955a, 1955b; Padykula and Gauthier, 1963) methods at the e l - ectron microscopical l e v e l . These methods are extended to i n - clude newborn kidneys. These are known to have d i f f e r e n t trans- port capacity and might therefore show differences i n ATPase a c t i v i t y , i f the two processes are related. Generally, the new- born kidney i s f u n c t i o n a l l y i n e f f i c i e n t and cannot withstand changes i n acid and base intake (Wacker et a l . , 1961; Moog, 1965), or changes i n hydration (Pinkstaff et a l . , 1962; Wach- stein and Bradshaw, 1965). Rates of glomerular f i l t r a t i o n and tubular absorption are low when compared with those of the adult kidney. The modifiers of enzymatic a c t i v i t y , p-hydroxymercuri- benzoate (PHMB) and L-cysteine, are used i n an i n i t i a l e f f o r t towards determining the s p e c i f i c i t y of the enzymatic a c t i v i t y demonstrated. Although the s p e c i f i c i t y of the enzyme system i s not f u l l y and thoroughly investigated, i t w i l l be designated as ATPas~e i n the following report. I I MATERIALS AND METHODS A. MATERIALS Kidneys were obtained from a d u l t , 3 day-old, 24 hour o l d , 12 hour-old, 2 hour-old and 1 hour-old r a t s . Adult r a t s were k i l l e d by an i n t r a - p e r i t o n e a l i n j e c t i o n of sodium pento- b a r b i t o l (0.6 cc of 330 mg. sodium pentobarbitol/10 ml. water) Newborn r a t s were k i l l e d by d e c a p i t a t i o n . The kidneys were r e - moved immediately and placed, i n a d i s h c o n t a i n i n g 5% g l u t a r a l - dehyde bu f f e r e d w i t h 0.1M sodium cacodylate ( S a b a t i n i et a l . , 1963). C o r t i c a l t i s s u e was cut wit h sharp r a z o r blades i n t o 1 mm. cubes and immersed i n the f i x a t i v e . ( A l l the procedures were c a r r i e d out at room temp- erature unless otherwise stated.) B. PRE-EIXATION IN GLUTARALDEHYDE To combine h i s t o c h e m i s t r y w i t h e l e c t r o n microscopy, i t was found that p r e - f i x a t i o n was of t e n necessary to adequate l y preserve the u l t r a s t r u c t u r a l f e a t u r e s o f the t i s s u e ( B a r r - n e t t , 1959; Holt and Hic k s , 1961). Without p r e - f i x a t i o n there was a l o s s of f i n e d e t a i l s and t h e r e f o r e i t was d i f f i c u l t to determine with which s t r u c t u r e s the products of the histochem- i c a l r e a c t i o n s were a s s o c i a t e d . Osmium t e t r o x i d e , the most common and u s e f u l f i x a t i v e then, gave e x c e l l e n t c y t o l o g i c a l f i x a t i o n but s e r i o u s l y reduced o r destroyed the a c t i v i t y of many enzymes (Holt and Hi c k s , 1961; S a b a t i n i et a l . , 1963). There was need f o r a f i x a t i v e which would r e t a i n s a t i s f a c t o r y u l t r a s t r u c t u r e as w e l l as preserve enough enzymatic a c t i v i t y to be demonstrable w i t h h i s t o c h e m i c a l techniques. Buffered formaldehyde was used by. H o l t and Hicks (1961). S a b a t i n i et a l . , (1963, 1964) introduced glutaraldehyde and other d i - aldehydes as s u i t a b l e f i x a t i v e s f o r u l t r a s t r u c t u r a l and cy t o - chemical s t u d i e s . Glutaraldehyde i s the most widely used at the present time. 8. Commercial glutaraldehyde preparations are rather crude and have to be p u r i f i e d by various means (Fahimi and Drochmans, 1968). In these experiments, the stock solution of 25?& glutaraldehyde (Eastman Organic Chemicals, D i s t i l l a t i o n Products Industries, Rochester 3, New York) was always f i l t e r - ed through activated charcoal (Fahimi and Drochmans, 1968) just p r i o r to use. As w i l l be discussed l a t e r , the duration of pre- f i x a t i o n i n 57° glutaraldehyde-cacodylate (0.1M) affected ATPase a c t i v i t y at pH 9.4 especially i n the kidneys of newborn r a t s . The times of f i x a t i o n were varied as indicated i n Table I. TABLE I AGE OF THE ANIMALS AND THE LENGTH QF FIXATION OF THE RESPECTIVE KIDNEYS Age of animals Length of f i x a t i o n (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 f i x a t i o n i n glutaraldehyde, the tissue blocks were washed i n the cacodylate buffer f o r at least 1 hour or stored i n buffer overnight (4° C) p r i o r to incubation. C. INCUBATION FOR HISTOCHEMISTRY Two general procedures were followed f o r the demon- st r a t i o n of ATPase a c t i v i t y i n 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. d i s t i l l e d water 20 ml. T r i s maleate buffer (pH 7.2) 3 ml. 2$ lead n i t r a t e (It was added gradually with constant s t i r r i n g to avoid pre c i p i t a t i o n . ) 5 ml. 0.1 M magnesium sulfate 25 mg. ATP (Sigma) The pH of the solution was adjusted to 7.2 i f necessary. It was f i l t e r e d and made up to 50 ml. (b) Preparation of incubating medium at pH 9.4 10 ml. 2#> sodium b a r b i t o l 5 ml. 2fo calcium chloride 15 ml. d i s t i l l e d water 0.15 mg. ATP (Sigma) was added to the above mixture, the pH adjusted to 9.4, f i l t e r e d and f i l l e d up to 50 ml. with d i s t i l l e d water. The solutions were always made up fresh and used immediately. Routinely, the tissue blocks were incubated i n the respective media at 37°C f o r 2 hours. Following incubation at pH 7.2, the tiss u e was rinsed with and stored i n buffer over- night at 4°C. Following incubation at pH 9.4, the tissue was washed i n several changes of ifo calcium chloride f o r \ hour and several changes of 2% cobalt n i t r a t e f o r 15 mins. before being kept i n buffer overnight at 4°C. 10. D. EFFECT OF MODIFIERS The response to modifiers (activators and in h i b i t o r s ) was tested using 2.5 x 10*"̂  M! L-cysteine (Sigma) as a source of s u l f h y d r y l groups, and 2.5 x 10"^ M PHMB (Sigma) as a mer- c u r i a l compound (Padykula and Herman, 1955b; Gauthier, 1967). PHMB i s a mercaptide forming agent and i n h i b i t s enzymes having s u l f h y d r y l groups at t h e i r active centres (Glenner, 1968). L-cysteine, an SH- compound, would activate an SH-dependent enzyme, while at the same time strongly i n h i b i t any al k a l i n e phosphatase a c t i v i t y (Padykula and Herman, 1955b). It i s be- lieved that a l k a l i n e phosphatase could act on ATP as a subst- rate (Padykula and Herman, 1955b; Freiman and Kaplan, 1959; P e r s i j n et a l . , 1961; Hori and Chang, 1963). It has been shown that any modifier should be used both before and during incubation to overcome the eff e c t s of " d i f f e r e n t i a l d i f f u s i o n of modifier and substrate" (Glenner, 1968). A pre-incubation exposure was effected by adding either L-cysteine or PHMB to the glutaraldehyde-cacodylate f i x a t i v e f o r the l a s t hour of f i x a t i o n . For comparison some tissue blocks were not pre-incubated with the modifiers i n the f i x a t e i v e . PHMB i s highly insoluble i f i t i s added d i r e c t l y to the incubating media, as has been suggested (Gauthier, 1967).' Therefore i t was f i r s t dissolved i n a d i l u t e a l k a l i n e solution of sodium hydroxide before being incorporated into the incub- a t i n g media or the f i x a t i v e . 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, a l l 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 i n buffer, f o r 1 hour i n 1$ osmium tetroxide buffered with 0.1 M sodium cacodylate. This increased the con- t r a s t of the membrane systems and s t a b i l i s e d the f i n e s t r u c t - ure of the c e l l s maintained by glutaraldehyde f o r Epon embedd- ing (Sabatini et a l . , 1963, 1964). G. ELECTRON MICROSOPPY The tissue blocks were dehydrated through a graded series of alcohol, h a l f and h a l f of absolute alcohol and propy- lene oxide, and propylene oxide alone. The tiss u e was then i n - f i l t r a t e d with a 1:1 mixture of propylene oxide and Epon f o r • 2 to 3 hours. Each tissu e block was embedded i n a g e l a t i n cap- sule containing fresh Epon, and the Epon allowed to polymerise overnight i n a 65°C oven. Sections were cut with glass knives on a Porter Blum MT-2 ultramicrotome. S i l v e r or gold sections were picked up on uncoated copper grids and examinedri^ri'th a P h i l i p s EM 200. A l l the sections were unstained. 12. I l l 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 l i g h t microscope (Maunsbach, 1966a, 1966b; Tisher et a l . , 1966; R o u i l l e r , 1969 - a review). V/ith the introduct- ion of the electron microscope, some of the older and c l a s s - - i c a l descriptions of renal structure have been confirmed and - also much extended (Pease, 1955; Yamada, 1955; Suzuki, 1958; Maunsbach et a l . , 1962; Porter and Bonneville, 1964; Bulger, 1965; Maunsbach, 1966a, 1966b; Tisher et a l . , 1966; G r i f f i t h et a l . , 1967; Latta et a l . , 1967; Ericsson and Trump, 1969; Simon and Chatelanat, 1969). Only a b r i e f description of the more prominent u l t r a s t r u c t u r a l features of the proximal tubule, d i s t - a l tubule and glomerulus w i l l be presented here. These charact- e r i s t i c s were the c r i t e r i a used f o r assigning the enzymatic r e - action f o r ATPase to a given portion of the nephron. To the naked eye, the kidney which has been fr e s h l y removed, i s a g l i s t e n i n g , reddish-coloured, bean-shaped s t r u c t - ure. It i s firm to the touch. A section through the kidney reveals a c l e a r demarcation between the reddish-brown cortex -and the paler medulla. Only the c o r t i c a l structures were studied i n t h i s present inve s t i g a t i o n . The proximal tubule (Pease, 1955; Porter and Bonn- e v i l l e , 1964; Bulger, 1965; Trump and Ericsson, 1965; Mauns- bach, 1966a, 1966b; Tisher et a l . , 1966; Latta et a l . , 1967; Ericsson and Trump, 1969) constitutes most of the cortex. The epithelium of the tubule consists of a single layer of trun- cated pyramidal c e l l s . In the a p i c a l portion of the c e l l s bordering the lumen, the plasma membrane i s thrown into folds to form numerous clos e l y packed m i c r o v i l l i . These constitute the brush border (Figures 1, 18-19, 22, 24, 37, 40). An 13. electron-opaque, PAS posit i v e layer of material, the glyco- calyx, covers the plasma membrane of the m i c r o v i l l i (Trump and Ericsson,,1965; Latta et a l . , 1967; Ericsson and Trump, 1969). Within the m i c r o v i l l i , a core of electron-dense-mater- i a l is- sometimes present. (Figure 19) Small tubular invaginat- ionssfrom the bases of the m i c r o v i l l i , v e s i c l e s and vacuoles of various sizes, are abundant i n the a p i c a l cytoplasm (Figures 1, 18-19, 22, 24, 40). It i s postulated that these invaginat- ions, v e s i c l e s and vacuoles represent one pathway fo r tubular reabsorption of larger molecules (Porter and Bonneville, 1964; Latta et a l . , 1967; Ericsson and Trump, 1969). In the b a s i l a r zone of the proximal tubule c e l l s , deep infoldings of the plasma membrane divide the cytoplasm i n - to numerous slender compartments within which mitochondria are enclosed. The processes from one c e l l i n t e r d i g i t a t e extensive- l y with processes from adjacent c e l l s (Figures 2, 18, 22-23, 40). As expected of these f u n c t i o n a l l y active c e l l s , a great number of mitochondria, cl o s e l y associated with the infolded membranes of the b a s i l a r processes, are present. These mitoch- ondria are large, elongated and possess numerous c r i s t a e . They are oriented perpendicular to the basement membrane (Figures 1, 23). At the l a t e r a l c e l l surface, there i s also extensive i n t e r - d i g i t a t i n g cytoplasmic processes. Some are small and are con- fined to the a p i c a l or b a s i l a r regions of the c e l l , whereas others extend from the apex to the base (Bulger, 1965). The brush border, basal and l a t e r a l i n t e r d i g i t a t i o n s greatly i n - crease the surface area of the c e l l s and therefore, also the number of s i t e s 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 d i s t a l tubule (Latta et a l . , , 1967; Ericsson and Trump, 1969).i s lower than that of the proximal tubule. The c e l l s are cuboidal i n shape. In the region of the nucleus, the c e l l s may bulge into the lumen. The a p i c a l plasma membrane i s not so highly d i f f e r e n t i a t e d s t r u c t u r a l l y 14. as i n the proximal tubule. There i s no d i s t i n c t brush border although frequently there are some small, short m i c r o v i l l i ( F i g - ure 38). A large number of v e s i c l e s may be seen i n the a p i c a l cytoplasm. A prominent c h a r a c t e r i s t i c are the numerous b a s i l a r processes containing large and slender mitochondria, which may extend almost to the luminal surface (Figures 3, 38). The f i n e structure of the glomerulus as seen under - the electron microscope was described as early as 1953 by H a l l . Since then there have been many more studies on the u l t r a s t r u c t - ure of the normal (Pease, 1955; Yamada, 1955; H a l l , 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 c a p i l l a r y endothelium and on thenother by the v i s c e r a l epithelium of Bowman's capsule (Figures 4, 20-21, 25, 39). The cytoplasm of the endothelium i s extremely attenuated. These cytoplasmic prolongations possess round pores or fenestrae. It i s only i n the region of the nuc l - eus that the endothelial c e l l projects into the c a p i l l a r y lumen. The basement membrane, interposed between the endo- thelium and the epithelium, forms, a continuous b a r r i e r i n the f i l t r a t i o n process. It i s 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 i s quite thick and prominent ( F i g - : ures 4, 21, 25, 39). The v i s c e r a l e p i t h e l i a l c e l l s of Bowman's capsule, or better known as the podocytes, are highly specialised. The c e l l s 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 c e l l i n t e r d i g i t a t e extensively with those of adjacent c e l l s thus g i v i n g r i s e to an i n t r i c a t e network. The foot processes from one podocyte may rest on the basement membrane of several c a p i l l a r i e s (Figure 4). Conversely, each c a p i l l a r y may receive contributions from more than one podocytic c e l l . Recently, i t 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 c e l l bodies (Jones, 1969). (b) Newborn kidney The fresh, unfixed kidney of a newborn rat i s small, pale and translucent. It i s soft to the touch and i t s * shape i s e a s i l y deformed by applying pressure. During the f i r s t few weeks of l i f e , the rat kidney grows through the formation of new nephrons i n the nephrogenic zone of the cortex. Its.?' weight i n - creases seven-fold within the f i r s t ten days (Wachstein and Brad- shaw, 1965). In sections of the kidney cortex, the tubules and glomeruli are not cl o s e l y packed together as i n the adult k i d - ney. Instead an abundant stroma of mesenchymal elements sep- arates the tubules from each other and from the glomeruli. Tub- ules i n various stages of d i f f e r e n t i a t i o n are always present within the same specimen at any one time (Clark, 1957; Suzuki,' 1958). This was found to be so even i n a 1 hour-old kidney. Some c o r t i c a l tubules appear r e l a t i v e l y undifferent- iated (Figures 5-7, 11-12, 26-28, 34-35, 41). The c e l l s are low and a large nucleus occupies most of the c e l l 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 di s t r i b u t e d throughout the cytoplasm. The mitochondria possess only a few c r i s t a e . The plasma membrane at the luminal surface i s simple i n contour or may be folded to form some short m i c r o v i l l i (Figures 6-7, 11- 12, 27-28). An occasional ci l i u m i s present (Figure 35). The b a s i l a r membrane too, i s not as complex as i n the adult with -few, i f any, i n t e r d i g i t a t i o n s . The l a t e r a l membrane between two adjacent c e l l s i s simple, or the beginnings of cytoplasmic i n t e r d i g i t a t i o n s may be observed (Figures 5, 26-27). At t h i s stage, i t does not seem possible to di s t i n g u i s h between the various types of tubules c h a r a c t e r i s t i c of each segment of the nephron. 16. In the process of renal c e l l u l a r d i f f e r e n t i a t i o n , the simple and primitive e p i t h e l i a l c e l l s are transformed into highly d i f f e r e n t i a t e d f u n c t i o n a l c e l l s . The successive stages - of t h i s process has not been f u l l y worked out yet (Du Bois, 1969). An e s s e n t i a l component of renal c e l l u l a r d i f f e r e n t i a t e ion i s the elaboration of c e l l membrane f o r the formation of the brush border i n the proximal tubules, and the basal and l a t e r a l i n t e r d i g i t a t i o n s i n both the proximal and d i s t a l tubules. Among the d i f f e r e n t i a t i n g tubules, the developing proximal tubule (Figures 8, 1 3 - 1 5 , 3 0 - 3 2 , 41, 44) i s most e a s i l y ident- i f i a b l e by the presence of long, slender m i c r o v i l l i which may s t i l l be sparse or may be cl o s e l y packed together. The brush border i s acquired through the progressive accumulation of ap- i c a l m i c r o v i l l i (Clark, 1957; Suzuki, 1958; Du Bois, 1969). It was observed that where a brush border was prominent, a p i c a l v e s i c l e s and vacuoles as well as a number of electron-dense granules were also present (Figures 8, 14-15, 30-32). There i s an increase i n the number of basal and l a t e r a l cytoplasmic i n t e r - d i g i t a t i o n s (Figures 8, 31-32). Simultaneously the mitochondria become regularly aligned perpendicular to the basement membrane within the b a s i l a r cytoplasmic compartments. Clark (1957) views the formation of the b a s i l a r infoldings as a res u l t of progressive f l u t i n g s of the c e l l membrane. Suzuki (1958) how- ever, hypothesises that v e s i c l e s gather around the b a s i l a r portions of the c e l l s and around the mitochondria. The v e s i c l e s coalesce to form small, flattened sacs which i n turn come t o - gether as larger sacs wrapping around the mitochondria. By t h i s process, the b a s i l a r cytoplasmic compartments are formed. As with the c o r t i c a l tubules, glomeruli in many stages of development are present within the same specimen (Figures 9-10, 16-17, 33, 36, 43). Some glomeruli are s t r u c t - ' u r a l l y immature (Figures 9, 16-17, 36)while others have the form of adult glomeruli although smaller i n size (Figures 10, 33, 43). Du Bois (1969) describes the progressive stages of development of the glomerulus i n the embryonic kidney. In i t s e a r l i e s t form, a mass of podocytic c e l l s , prismatic i n shape 17. (Figures 9, 16, 36), denotes the region of the glomerulus. Further along i n development, the a p i c a l pole of the podocytic c e l l containing the nucleus bulges out into Bowman's space, while the basal pole sends out cytoplasmic prolongations which d i f f e r e n t i a t e into the foot processes (Figures 17, 33, 43). These increase i n length and complexity and come to l i e on the t r i l a m i n a r 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 c a p i l l a r i e s (Eigure 10). B. EFFECT OF FIXATION ON ULTRASTRUCTURB (a) Adult kidney As early as 1955, Pease observed that small d i f f - erences i n the preparation techniques when applied to the k i d - ney, could cause, large v a r i a t i o n s i n the morphology of the kidney tubular elements. Since then h i s observations have been substantiated (Maunsbach et a l . , 1962; Trump and Ericsson, 1965; "Maunsbach, 1966a). The a p i c a l ends of the c e l l s are es p e c i a l l y l a b i l e . The proximal tubule c e l l s show great s e n s i t i v i t y to f i x a t i o n conditions and are affected both by the character of the f i x a t i v e and by the method of application of the f i x a t i v e s olution. In the present study, c o r t i c a l t i s s u e from the ex- cised kidney was fixed by the immersion of small blocks i n the f i x a t i v e solution consisting of 5f° glutaraldehyde buffered with 0.1 M sodium cacodylate. In general, the f i x a t i v e used gave adequate preservation of the organelles i n the cytoplasm. However, by t h i s method of immersion i n the f i x a t i v e , some •a r t i f a c t s are present i n the proximal tubules (Maunsbach et a l . , 1962; Maunsbach, 1966a; Tisher et a l . , 1966). The lumens •of the proximal tubules were more often than not closed r e s u l t i n g i n a region of c l o s e l y packed m i c r o v i l l i (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 a r t i f a c t u a l r e s u l t i n g from excessive swelling of the c e l l s during f i x a t i o n (Maunsbach, 1966a). There may be some c e l l - u l a r debris i n the lumens of the tubules. These take the form of cytoplasmic b i t s and pieces, or even whole, less osmiophilic c e l l s that seem to be extruded into the lumen. An occasional tubule was not collapsed but possessed a patent lumen. Tisher et a l . (1966) interprets t h i s as due to dehydration i n the preparative techniques f o r electron microscopy following c e l l swelling during f i x a t i o n . Ocaasionally the membranes i n the b a s i l a r part of the c e l l s were separated g i v i n g r i s e to extra- c e l l u l a r compartments of d i f f e r e n t s i z e s . The extent of the presence of e x t r a c e l l u l a r compartments was not consistent ( F i g - ures 2-3, 18, 22-23, 38, 40). Variations between c e l l s i n the same tubule as well as v a r i a t i o n s between d i f f e r e n t tubules were observed. These e x t r a c e l l u l a r compartments are probably indications of sensitive c e l l u l a r reactions to p h y s i o l o g i c a l and pathological ( i n t h i s case, f i x a t i o n ) changes i n the en- vironment . The d i s t a l tubules and the glomeruli were more res i s t a n t to the e f f e c t s of f i x a t i o n and were always morphol- T o g i c a l l y well preserved. The lumens of the d i s t a l tubules were open. There could have been some c e l l swelling! r e s u l t i n g i n a reduction i n size of the lumen but t h i s was not so obvious as i n the proximal tubules where the brush border accentuated the e f f e c t s . (b) Newborn kidney The same concentration of f i x a t i v e and a s i m i l a r method of f i x a t i o n , that i s , by the immersion of small t i s s u e blocks, was applied to the newborn kidney. The e f f e c t s of f i x a t i o n varied depending on the stage of d i f f e r e n t i a t i o n of the tubules. The morphologically undifferentiated c o r t i c a l tubular c e l l s consisted of a nucleus, some cytoplasm and a few organelles, primarily small, round mitochondria with 19. sparse c r i s t a e , scattered throughout the cytoplasm. The mito- chondria i n the immature c e l l s seemed more susceptible to - f i x a t i o n a r t i f a c t s as compared with the adult. Quite a number of the mitochondria were "exploded". (Figures 27-28, 30-31, 38) Otherwise, the preservation of u l t r a s t r u c t u r e was generally good. The 1tubules had wide open lumens often f i l l e d with c e l l - u l a r debris. Debris was also found i n the extratubular stroma. There appeared to be more c e l l u l a r debris associated with the newborn kidney than with the adult kidney. In cross-sections of some undifferentiated tubules, a l l the c e l l s but one, were well preserved. This one c e l l was completely disintegrated with a nucleus that was swollen to immense proportions (Figure 29). Such a phenomenon was not observed i n adult kidney t i s s u e . Developing proximal tubules were recognised by the presence of long, slender m i c r o v i l l i i n the a p i c a l surface membrane (Figures 8, 13-15, 30-32, 38). They may be few i n number or may be c l o s e l y packed to form a d i s t i n c t brush border. In the tubules with few m i c r o v i l l i , the lumens were open but contained some c e l l u l a r debris. In more advanced proximal tub- ules, that i s , those having a well-developed brush border and a number of v e s i c l e s and vacuoles i n the a p i c a l cytoplasm, the lumens were closed (Figures 8, 15, 31-32). Less osmiophilic c e l l s were often seen being extruded into the lumen (Figure 8). The response of these tubules to f i x a t i o n was very s i m i l a r to that of the adult. The mitochondria were generally s t i l l small and showed some a r t i f a c t s of f i x a t i o n . The glomeruli were always adequately preserved u l t r a - s t r u c t u r a l l y independent of the stage of development. G. EFFECTS OF FIXATION ON ENZYMATIC ACTIVITY The histochemical l o c a l i s a t i o n of ATPase enzymatic a c t i v i t y 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 f i x a t i o n (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 I T - - - 1 hour 1 - - - 1 hour JL 2 - - - 1 hour JL 4 + + + Fixative; 5f° glutaraldehyde i n 0.1M sodium cacodylate buffer at pH 7.2. + reaction p r e c i p i t a t e present reaction p r e c i p i t a t e absent p proximal tubules d d i s t a l tubules g glomeruli -NB. In newborn tissue, wherever i t was not possible to d i f f e r - entiate between a developing proximal tubule and a develop- ing d i s t a l tubule, i t was assumed that the undifferentiated tubules represented both types. 21. i e r , 1963) reported that>similar r e s u l t s were obtained with e i t h e r method (Novikoff et a l . , 1961). In the adult kidney, these observations on plasma membrane ATPase a c t i v i t y was found to be so. However, with immature newborn kidneys i t was ob- served that the length of f i x a t i o n affected the l o c a l i s a t i o n of membrane ATPase a c t i v i t y d i f f e r e n t l y depending on the method that was employed (See Table I I ) . The time of f i x a t i o n was not important when deal- ing with adult kidney t i s s u e . Even up to 5 hours f i x a t i o n time there was much reaction p r e c i p i t a t e associated with the membranes, using either the lead method at pH 7.2, or the calcium method at pH 9 . 4 . The time of f i x a t i o n however, was more c r i t i c a l i n the case of immature newborn kidneys, i f the l o c a l i s a t - ion f o r enzymatic a c t i v i t y was carried out at pH 9 . 4 . A 2 hour- old kidney a f t e r 2 hours in the f i x a t i v e solution s t i l l showed reaction product f o r the enzyme reaction at pH 7.2. But l i t t l e or no p r e c i p i t a t e was present at pH 9 . 4 with a s i m i l a r time of f i x a t i o n . It seemed that the more immature the kidney i n terms of age postnatally, the shorter the time of f i x a t i o n needed to preserve enough enzymatic a c t i v i t y to be demonst- rable at pH 9 . 4 with the calcium method. Adequate amounts of enzymatic a c t i v i t y were always present to r e s u l t i n a p o s i t i v e reaction at pH 7.2. With the kidney of a 1 hour-old rat, even a § hour of f i x a t i o n was s u f f i c i e n t to i n h i b i t any enzymatic a c t i v i t y at pH 9 . 4 that might have been present i n the t i s s u e . With the f i x a t i v e used ( 5 $ glutaraldehyde i n 0.1M sodium cacodylate) good preservation of u l t r a s t r u c t u r e had to be s a c r i f i c e d to prevent complete i n h i b i t i o n 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 f i n a l product of the ATPase enzymatic reaction was v i s u a l i s e d as electron-dense 22. p a r t i c l e s , which may be i n the form of f i n e granules or i n the form of larger aggregates. The s i t e s of deposition of the reaction product would indicate areas of enzymatic a c t i v i t y . The proximal tubules showed two d i s t i n c t regions of enzymatic a c t i v i t y , the m i c r o v i l l i of the brush border and the i n t e r d i g i t a t i o n s of the basal and l a t e r a l membranes (Figures 1-2). The p r e c i p i t a t e was most often located on the outer sur- face of the plasma membranes of the m i c r o v i l l i . At times, but rather r a r e l y , p r e c i p i t a t e was associated with the inner cyto- plasmic surfaces of the m i c r o v i l l i membranes. Pre c i p i t a t e could sometimes be demonstrated within some of the tubular invagin- ations a r i s i n g from the bases of the m i c r o v i l l i , and within some of the small a p i c a l v e s i c l e s and large a p i c a l vacuoles. Some of the membrane i n t e r d i g i t a t i o n s showed such an abundant deposition of reaction product that the space between the membranes was completely f i l l e d with p r e c i p i t a t e . Where the reaction was less intense, the pr e c i p i t a t e was found to be on the membrane i t s e l f and not free i n the e x t r a c e l l u l a r space (Figure 2). However, the reaction p r e c i p i t a t e 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 p r e c i p i t a t e (Figure 2). Variations i n staining i n t e n s i t y of the c e l l s within the same tubule, as wel l as between d i f f e r e n t tubules was encountered. At times two tubules adjacent to each other, reacted d i f f e r e n t l y . One tubule showed reaction p r e c i p i t a t e i n the brush border and b a s i l a r i n foldings while the other showed l i t t l e or no react- ion p r e c i p i t a t e . In the d i s t a l tubules the reaction product was confined mainly to the c e l l membranes of the b a s i l a r i n t e r - d i g i t a t i n g cytoplasmic processes and the l a t e r a l membranes separating two adjacent c e l l s (Figure 3). As i n the case of the proximal tubules, p r e c i p i t a t e may be found on both the e x t r a c e l l u l a r and cytoplasmic aspects of the i n t e r d i g i t a t i n g membranes and within the cytoplasm (Figure 3). Occasionally, the few short m i c r o v i l l i reacted p o s i t i v e l y and pr e c i p i t a t e 23. was seen on the m i c r o v i l l i membranes. The glomeruli were s i t e s of active enzymatic a c t i v - i t y (Figure 4). Abundant pr e c i p i t a t e was present both on the membranes and within the cytoplasm of the foot processes, i n the trilaminar, basement membrane but not i n the endothelium (Figure 4). (h) pH 9.4 - calcium method The r e s u l t s from the l o c a l i s a t i o n of ATPase act- i v i t y with the calcium method at pH 9.4 were s i m i l a r to those obtained with the lead method at pH 7.2. Enzymatic a c t i v i t y was demonstrated i n the brush border of the proximal tubules, i n the l a t e r a l and basal i n t e r d i g i t a t i o n s of the proximal and d i s t a l tubules, and i n the glomerular e p i t h e l i a l c e l l s . E. ENZYMATIC ACTIVITY OF THE NEWBORN KIDNEY (a) pH 7.2 - lead method (Figures 5-10) With t h i s method i t was possible to l o c a l i s e enzy- matic a c t i v i t y i n the kidneys of newborn rats of various ages, ranging from a 2 hqur-old to a 3.day-old. The time of pre- f i x a t i o n i n ~fo glutaraldehyde-cacodylate did not appear to af f e c t the l e v e l of enzymatic a c t i v i t y . In a very immature kidney, f o r example from a 2 hour- old rat, most of the tubules were s t i l l undifferentiated and i t was not possible to d i s t i n g u i s h between a proximal tubule and a d i s t a l tubule (Figures 5-7). A large number of these undifferentiated tubules reacted intensely when incubated i n the lead-ATP medium. The l a t e r a l membranes between neighbour- ing c e l l s , which were more often simple i n contour, were f i l l - ed with the reaction product. In fa c t , the boundaries between i n d i v i d u a l c e l l s i n the tubules were c l e a r l y outlined by the deposition of the prec i p i t a t e i n the l a t e r a l membranes ( F i g - • ures 5-7). Some pre c i p i t a t e was found on the plasma membranes l i n i n g the lumens of the tubules (Figure 6). Some of the m i c r o v i l l i that were present reacted p o s i t i v e l y while others reacted negatively i n the incubating medium. At t h i s early 24. stage of d i f f e r e n t i a t i o n , the basal membranes did not show- any elaborate infoldings as seen i n the adult kidney (Figures 5 - 7 ) . There was usually no pr e c i p i t a t e on the basal membranes (Figures 5-6). Sometimes, however, a single c e l l within a tubule showed abundant pre c i p i t a t e on the basal membranes (Figures 5 , 7 ) . This was an exception. Besides the undifferentiated tubules, tubules i n various stages of d i f f e r e n t i a t i o n were also present within the newborn kidney. A developing proximal tubule possessed a f a i r - l y well-developed brush border and many more i n t e r d i g i t a t i o n s of the basal and l a t e r a l membranes (Figure 8). These i n t e r - d i g i t a t i o n s were s t i l l by no means as extensive as i n the adult. Reaction p r e c i p i t a t e was found encrusting the plasma membranes of the m i c r o v i l l i i n the brush border (Figure 8). Occasionally some pr e c i p i t a t e was present within the micro- v i l l i . The basal infoldings had fin e p r e c i p i t a t e adhering to the, membranes. Usually the space between the membranes was not f i l l e d up with p r e c i p i t a t e as was the case with the un- d i f f e r e n t i a t e d tubules (Figure 8). , . . Developing d i s t a l tubules showed an increase i n the number of short, i r r e g u l a r l y shaped m i c r o v i l l i . The r e - action p r e c i p i t a t e generally coated the membranes of the micro- v i l l i but was also seen within the cytoplasm of the m i c r o v i l l i . The l a t e r a l and basal i n t e r d i g i t a t i o n s were frequently so packed with p r e c i p i t a t e that i t was impossible to t e l l i f the p r e c i p i t a t e 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 i n the kidney, im- mature undifferentiated glomeruli were present together with more well-developed glomeruli within the same specimen. In the undifferentiated glomerulus, the podocytic c e l l s were close l y packed so that t h e i r l i m i t i n g membranes were quite often i n apposition (Figure 9). Some podocytes showed the beginnings of cytoplasmic prolongations which would eventually develop to form the foot processes. It was observed that r e - 25. action product was almost always present where two sets of membranes were apposed.(Figure 9). Portions of the membranes of the podocytic c e l l which was not close to the membranes of another podocytic c e l l sometimes showed pr e c i p i t a t e but t h i s was not usually so. Not a l l the c e l l s i n the same glom- erulus were reactive. Some c e l l s showed no detectable enzy- matic a c t i v i t y . In a glomerulus further along the course of d i f f e r - e n tiation, the foot processes of the podocytic c e l l s had i n - creased i n number and formed a complex network along the ; t r i - laminar basement membrane.. The cytoplasm of the endothelial c e l l was becoming attenuated and fenestrated but not to sucM a great extent as i n the adult (Figure 10) Reaction p r e c i p i t - ate was found on the membranes as well as within the cyto- plasm of the foot processes and podocytic c e l l body, which was s t i l l situated quite near to the c a p i l l a r i e s . The endo- t h e l i a l c e l l s were usually non-reactive. I f p r e c i p i t a t e were present, i t was found mainly on the cytoplasmic surface of the membranes and i n the cytoplasm (Figure 10). (b) pH 9.4 - calcium method (Figures 11-17) FormATPase enzymatic a c t i v i t y i n a newborn kidney to be demonstrable with t h i s method, the time of p r e - f i x a t i o n i n glutaraldehyde was found to be c r i t i c a l (Refer to Table I I ) . A 3 day-old kidney fixed f o r 5 hours p r i o r to i n - cubation showed enzymatic a c t i v i t y i n the proximal and d i s t a l tubules but not i n the glomerulus. In the proximal tubule, p r e c i p i t a t e was found a l l along the infoldings and also coat- ing the m i c r o v i l l i . Quite a number of the a p i c a l tubular i n - vaginations from the bases of the m i c r o v i l l i were lined with the f i n e p r e c i p i t a t e (Figure 15). The lateral'and basal i n t e r - d i g i t a t i o n s of the d i s t a l tubule gave positive r e s u l t s . A 24 hour-aid kidney pre-fixed f o r 5 hours showed no reaction p r e c i p i t a t e . But i f the time of f i x a t i o n was shortened to 3 hours, there were indications of enzymatic a c t i v i t y i n the tubules. In the glomerulus, reaction product 26. was sometimes present and sometimes not. I f present, it-was l o c a l i s e d on the plasma membranes of the podocytic processes. Thus by c o n t r o l l i n g the times of f i x a t i o n i t was possible to l o c a l i s e ATPase' enzymatic a c t i v i t y i n the tubules and glomeruli of the newborn kidney. In cases where there was just minimal a c t i v i t y , f o r example, a 1 2 hour-old kidney with 4 hours pre-fixation, or a 2 hour-old kidney with 2 hours pre - f i x a t i o n , or sometimes a 1 hour-old kidney with 1 hour pre-fixation, reaction p r e c i p i t a t e was observed mostly i n the tubular elements. The pr e c i p i t a t e , i n the form of discrete e l - ectron-dense p a r t i c l e s , were found on the membranes of the few m i c r o v i l l i and sometimes on the l a t e r a l membranes (Figures 1 1 - 1 2 ) . Where the reaction was more intense, f o r example, a 1 2 hour-old kidney with 2 hours pre-fixation, or a 1 hour-old kidney with \ hour pre-fixation, the s i t e s f o r the deposition of the reaction product f o r ATPase at pH 9 . 4 were si m i l a r to those at pH 7 . 2 (Figures 1 3 - 1 4 ) . Some tubules reacted p o s i t i v e - l y while others reacted negatively. This was not dependent on the degree of maturation of the tubules. In the immature tub- ules, the precipitate i n the l a t e r a l membranes c l e a r l y separ- ated one c e l l from the next. Some pre c i p i t a t e was also present on the r e l a t i v e l y simple plasma membrane l i n i n g the luminal surface. The brush border of the developing proximal tubules showed varying amounts of p r e c i p i t a t e . Some of the a p i c a l v e s i c l e s and vacuoles had reaction product- on t h e i r l i m i t i n g membranes (Figures 1 4 - 1 5 ) . In developing d i s t a l tubules, r e - action p r e c i p i t a t e was observed mainly i n the l a t e r a l memb- ranes even though some basal infoldings and some m i c r o v i l l i were present. Some of the tubules showed pr e c i p i t a t e i n -the region of the basement membrane but t h i s did not appear to be associated with the basal membranes. Observations on the enzymatic a c t i v i t y of the glomerulus at pH 9 . 4 were si m i l a r to those at pH 7 . 2 (Figures 1 6 - 1 7 ) . Glomerular enzymatic- a c t i v i t y appeared to be moressusceptible to f i x a t i o n e f f e c t s . With longer f i x a t i o n times, enzymatic a c t i v i t y i n the glom- erulus was in h i b i t e d 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 p r e c i p i t a t e present - reaction p r e c i p i t a t e absent p proximal tubules d. d i s t a l 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 a c t i v i t y was l o c a l i s e d mainly at the proximal tubules (Figures 18-19) and glomeruli (Figures 20-21). Within the proximal tubules, the preci p i t a t e was 28. present i n the brush border as we l l as i n the ' i n t e r d i g i t a t - ions (Figure 18). In the brush border the pr e c i p i t a t e was us- • u a l l y found uniformly d i s t r i b u t e d over the membranes of a l l the m i c r o v i l l i , but were at times clumped together i n certain regions within the brush border (Figure 19). This was i n t e r - preted as an a r t i f a c t rather than as an in d i c a t i o n of hetero- geneity of s e n s i t i v i t y to the e f f e c t s of PHMB among the 'micro- v i l l i . Variations i n the i n t e n s i t y of reaction at the i n f o l d - ings occured between i n d i v i d u a l c e l l s and between d i f f e r e n t tubules (Figure 18). Only the membranes of the podocytic foot processes showed an accumulation of the reaction prod- uct. There was no pre c i p i t a t e i n the cytoplasm of these c e l l s as was often noticed with tissue incubated without PHMB. There was also no prec i p i t a t e i n the basement membrane or endo- thelium (Figures 20-21). At pH 9-4, enzymatic a c t i v i t y 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 d i s t a l tubules showed no reaction product. There was an occasional d i s t a l tubule with p r e c i p i t a t e i n the i n t e r d i g i t a t i o n s . However, i n 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 pre c i p i t a t e i n the cytoplasm and on the membranes of the podocytic foot processes but not i n the base- ment membrane or endothelium (Figure 25). With pre-incubation, most of the glomeruli showed no reaction product. This observ- ation substantiated Glenner 1s (1968) suggestion that any modifier should be used both before and during incubation. This would ensure that the modifier had reached the s i t e of enzymatic a c t i v i t y p r i o r to incubation i n the substrate medium. L-cysteine had no effect on the l o c a l i s a t i o n of enzymatic a c t i v i t y at pH 7.2. But at pH 9 . 4 , a pre-incubat- ion exposure to L-cysteine in h i b i t e d p r a c t i c a l l y a l l enzy- matic a c t i v i t y i n the glomeruli and i n most of the d i s t a l 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 p r e c i p i t a t e present reaction p r e c i p i t a t e 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 eff e c t s on the enzymatic a c t i v i t y of the d i f f e r e n t i a t i n g tubules (Figures 26-32, 34-35) and glomeruli (Figures 33, 36). Most of the tubules showed intense reaction i n the l a t - e r a l membranes between c e l l s (Figures 26-27, 30-32, 34-35). The luminal surface membrane and m i c r o v i l l i , i f developed, show- ed v a r i a t i o n s i n the amount of pre c i p i t a t e (Figures 30-32). 30. In developing proximal tubules, the brush border was a d i s t - i n c t l y active enzymatic s i t e (Figures 30-32). The glomerulus had pr e c i p i t a t e associated es p e c i a l l y with membranes which were i n apposition (Figures 33, 36). At pH 9«4, no enzymatic a c t i v i t y was detected i n the newborn kidney when PELT-IB and L-cysteine were used. How- ever, these negative r e s u l t s cannot be considered too s i g - n i f i c a n t at the-present time. With a newborn kidney a couple of hours old, the i n h i b i t o r y e f f e c t s of the f i x a t i v e used were found to be so overwhelming that i t was impossible to assess the eff e c t s of the modifiers. G. CONTROLS In a l l the experiments, controls were run simult- aneously with the experimental specimens. The controls d i f f e r - - -ed only i n that ATP was omitted from the incubating media. With the calcium method at pH 9.4, no prec i p i t a t e was found i n either adult or newborn t i s s u e . With the lead method at pH 7.2, there was a tendency f o r some p r e c i p i t a t i o n i n some c e l l s (Figures 40, 44) but not i n others (Figures 37-39, 41-43). In adult kidney tissue, t h i s took the form of f i n e s t i p p l i n g of the cytoplasm, or association of the pre c i p i t a t e with the basement membrane or m i c r o v i l l i of the brush border (Figure 40). In the newborn kidney, a fin e d i f f u s e p r e c i p i t a t e was sometimes present. I f the m i c r o v i l l i were long and slender, as i n the developing proximal tubule, there was some prec i p i t a t e on the membranes (Figure 44). • These observations were not consistently present in a l l the specimens. I f prec i p i t a t e was present i n the con t r o l specimens, i t was always less than i n the experiment- a l s i t u a t i o n . H. NUCLEAR STAINING In both experimental and con t r o l specimens, nuclear s t a i n i n g was found to be e r r a t i c . Variations occured between 31. i n d i v i d u a l c e l l s i n the tubules and i n the glomeruli. Some n u c l e i were perfectly free of prec i p i t a t e while others show- ed a large amount. Nuclei from two adjacent c e l l s often be- haved d i f f e r e n t l y , with pr e c i p i t a t e present i n the one but not i n the other. Wherever pr e c i p i t a t e was observed, i t was mainly associated with the nucleolus and with the denser heterochrom- a t i n (Figures 10, 27). Even then there were differences i n i n t e n s i t y . 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) f o r the h i s t o - chemical l o c a l i s a t i o n of ATPase a c t i v i t y have been used widely to demonstrate enzymatic a c t i v i t y i n various tissues where active transport i s known to occur. Recently, the s p e c i f i c i t y of the histochemical l o c a l i s a t i o n of ATPase enzymatic a c t i v i t y with the Wachstein-Meisel procedure has been seriously quest- ioned (Moses et a l . , 1966; Rosenthal et a l . , 1966; Moses and Rosenthal, 1967, 1968; Rosenthal et a l . , 1969). The p o s s i b i l i t y that there i s non-enzymatic hydrolysis of the substrate ATP by the lead i n the incubating medium was proposed. It i s thought that t h i s non-enzymatic hydrolysis of ATP by lead could account f o r the deposition and l o c a l i s a t i o n of p r e c i - p i t a t e on plasma membranes, the most often observed s i t e s of intense reaction with the lead method. Moses arid Rosenthal (1968) suggest that there i s "a selective a f f i n i t y of certain t i s s u e - r e a c t i v e groups at the s i t e s of l o c a l i s a t i o n f o r the complexes formed by the i n t e r a c t i o n of lead and ATP." At the present time i t i s d i f f i c u l t to envision how t h i s tissue f a c t o r with a s e l e c t i v e a f f i n i t y f o r the products of the non-enzymatic hydrolysis of ATP could account f o r the substrate s p e c i f i c i t i e s of many plasma membranes (Novikoff, 1967), the e f f e c t s of modifiers only on some membranes (Novikoff, 1967) and the d i f f e r i n g patterns of s i t e s of l o c a l i s a t i o n of precipitate obtained with the same tissue under varying conditions. Mar- chesi (1968) i n h i s investigations of ATPase a c t i v i t y on red • blood c e l l membranes found that there was non-enzymatic hydrolysis of ATP i n the incubating medium but that i t did not account f o r the deposition of the lead phosphate on the red blood c e l l membranes. Jacobsen and Jorgensen (1969) found 33. the s t a i n i n g of the plasma membranes of the kidney character- i s t i c of enzymatic hydrolysis rather than non-enzymatic hydro- l y s i s , while Grossman and Heitkamp (1968) found no measurable non-enzymatic hydrolysis of ATP by lead i n chemical assays of the media used f o r the histochemical l o c a l i s a t i o n of ATPase a c t i v i t y . We are. f a r from an understanding of the complexities of the reactions involved i n the reaction mixtures used i n histochemistry, e s p e c i a l l y where a heavy metal l i k e lead i s present. It i s probable that the various constituents of the incubating media interact with each other. There i s a suggest- ion that lead i n the incubating media may not be i n 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 p r e c i p i t a t e on tissue sections may not be just simple lead phosphate but may be a more complex compound (Marchesi, 1968; Moses and Rosenthal, 1968). It i s e s s e n t i a l that variable of the reaction be c a r e f u l l y controlled. In the present study of ATPase enzymatic a c t i v i t y i n both the adult and newborn kidney ti s s u e , the composition of the incubating media were kept constant so that the significance of any differences i n the s i t e s of deposition of the reaction p r e c i p i t a t e could be assessed. Also a method not involving the use of lead s a l t s was deemed desirable as a comparison (Goldfischer et a l . , 1964) Therefore both the lead and calcium methods were applied i n the investigation of ATPase enzymatic a c t i v i t y i n adult and newborn kidney t i s s u e . Under the conditions employed i n t h i s present - study, only plasma membrane ATPase was demonstrable. Mito- chondrial ATPase was not detected although there would be an occasional mitochondrion with reaction p r e c i p i t a t e associated with i t . F ixation i n glutaraldehyde has been found to have an i n h i b i t o r y effect on mitochondrial ATPase (Torack and Barrnett, 1963; Lazarus and Barden, 1964; Wachstein and Besen, 1964; Essner et a l . , 1965; Vethamany and Lazarus, 1967; Anderson, 34. 1968). Fresh unfixed tissues.are of course i d e a l f o r demon- s t r a t i n g any ATPase a c t i v i t y , including mitochondrial ATPase, but t h i s i s not always possible at the l e v e l of the electron microscope. Fixation i n formalin preserves mitochondrial ATP- ase enzymatic a c t i v i t y i n some tissues (Lazarus and Barden, 1962; Wachstein and Bradshaw, 1962; Ashworth et a l . , 1963; Bradshaw et a l . , 1963; Otero-Vilardebo et a l . , 1963; Essner et a l . , 1965; Lazarus and Vethamany, 1965; Rechardt and Kokko, 1967; Vethamany and Lazarus, 1967;0gawa and Mayahara, 1969) but not i n others (Wachstein et a l . , I960; Wachstein and: Brad- shaw, 1962; Barden and Lazarus, 1963; Otero-Vilardebo et a l . , 1963; Wachstein and Besen, 1964; G-authier, 1967). It appears that mitochondria from d i f f e r e n t tissues show d i f f e r e n t sus- c e p t i b i l i t i e s towards f i x a t i o n . Where mitochondrial ATPase has been l o c a l i s e d , i t i s s t i l l not f u l l y agreed upon whether the p r e c i p i t a t e i s on the inner c r i s t a l membranes (Ashworth et a l . , 1963; Otero-Vilardebo et a l . , 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 d i f f e r e n t tissues may have d i f f e r e n t a f f i n i t i e s f o r lead s a l t s (Wilson, 1969)• There are three possible explanations to bear i n mind when considering the l o c a l i s a t i o n of reaction p r e c i p i t a t e • at the plasma membranes of kidney tubular c e l l s and glomerular c e l l s . F i r s t l y , f i x a t i o n of the kidney tissue i n glutaralde- hyde could a l t e r the c e l l membranes i n such a way that they act as b a r r i e r s to substances entering the c e l l s . This being the case, enzymes present within the cytoplasm and i n the c e l l organelles would d i f f u s e towards the membranes and react with the substrate i n the incubation medium, that i s ATP, thereby releasing the reaction precipitate at the e x t r a c e l l u l a r aspect of the c e l l membranes. This seems u n l i k e l y i n view of the experimental r e s u l t s presented here. Although reaction 35. p r e c i p i t a t e i s most frequently observed on the e x t r a c e l l u l a r aspects of the plasma membranes (Figures 2, 5-9, 11-18, 20- 23, 26-27, 30-36), i t i s . a l s o present within the cytoplasm (Figures 1, 3, 4, 10, 25, 36), i n the n u c l e i (Figures 10, 27) and occasionally within the mitochondria. It appears that substances from the substrate medium can enter the c e l l s . It i s e n t i r e l y possible that f i x a t i o n has altered the membranes in some way. Secondly, i n undifferentiated tubules of the new- born kidney intense reaction p r e c i p i t a t e i s present on the l a t e r a l membranes between i n d i v i d u a l c e l l s . In the glomeruli, apposed membranes show an accumulation of reaction product. It i s suggested that the enzymes on the luminal surface memb- ranes and basal membranes of the tubular c e l l s , and on the free membranes of the podocytic c e l l s may not be so firmly attached to the membranes and are therefore "washed" towards the apposed membranes. I f t h i s were the case, then one would expect gradients i n the i n t e n s i t y of the deposition of the reaction product..This was not observed. Occasionally, there would be intense reaction p r e c i p i t a t e 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 l o c a l i s a t i o n of reaction p r e c i p i t a t e on the membranes could indicate ATPase enzymatic a c t i v i t y at the membranes themselves, as suggested here. In view of the above, i t seems most l i k e l y that a c t u a l plasma membrane enzy- matic a c t i v i t y was demonstrated i n the present experiments. B. PLASMA MEMBRANE ENZYMATIC ACTIVITY (a) Adult kidney With both the lead and calcium methods f o r the histochemical l o c a l i s a t i o n of ATPase enzymatic a c t i v i t y , the reaction p r e c i p i t a t e was deposited on the plasma memb- ranes of the proximal and d i s t a l tubules and glomeruli. Variations i n in t e n s i t y of staining were present. Besides, some tubules and glomeruli showed the presence of the react- 36. ion product while others did not. These s i m i l a r observations had been made previously by Wachstein and Besen (1964). This v a r i a b i l i t y has been interpreted as a r t i f a c t u a l (Goldfischer et a l . , 1964) although other possible explanations could also • be offered. At the present stage of development of histochem- i c a l techniques, quantitation of enzymatic a c t i v i t y can only be based on the density of the f i n a l reaction product deposit- ed (Glenner, 1965). Therefore v a r i a t i o n s i n sta i n i n g i n t e n s i t y found i n the adult kidney when incubated f o r an ATPase reaction would indicate various degrees of enzymatic a c t i v i t y . Different nephrons at any one time may be i n d i f f e r e n t f u n c t i o n a l states ( C a u l f i e l d and Trump, 1962). In the proximal tubules, d i f f e r - ences i n the i n t e n s i t y of the ATPase reaction could indicate differences i n enzymatic a c t i v i t y associated with the various segments. It has been found that proximal tubules show segment- ation i n terms of t h e i r u l t r a s t r u c t u r e , function and histochem- i c a l reactions (Kissane, 1961; Maunsbach, 1966b; Tisher et a l . , 1966; Jacobsen et a l . , 1967; Latta et a l . , 1967; Ericsson and Trump, 1969). In the present study, the differences i n 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- i c a l methods f o r the demonstration of enzymatic a c t i v i t y to the kidney, i s not only an attempt to l o c a l i s e the s i t e s of enzymatic a c t i v i t y i n terms of the ul t r a s t r u c t u r e of the tissue, but also to t r y to correlate s p e c i f i c s i t e s of enzymatic ac t - i v i t y with the known functions of the ti s s u e . Unfortunately f o r most enzymes i n the kidney, including ATPase, we have no s p e c i f i c idea of the ro l e they play i n kidney function. Most of the suggestions that have been put forward f o r the role s of ATPase i n kidney function are highly speculative. Such speculations, based on available data, are us e f u l and may lead to further experiments which might help elucidate some of the complexities of kidney function. Proximal tubules show l o c a l i s a t i o n of reaction product to the membranes of the brush border, the basal and 37. the l a t e r a l i n t e r d i g i t a t i o n s . The ATPase reaction i n these s i t e s was not eliminated by the addition of L-cysteine or PHMB to the incubating media i n d i c a t i n g 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 s u l f h y d r y l groups as we l l as an i n h i b i t o r of a l k a l - ine phosphatase, which enzyme has been found to be also pres- ent i n the brush border. In t h i s investigation L-cysteine had no ef f e c t on brush border enzymatic a c t i v i t y , i n agreement with the findings of Padykula and Herman (1955b) but not with the findings of Freiman and Kaplan (1959) where brush border'act- i v i t y was abolished by L-cysteine. This discrepancy i s probab- l y due to species differences (Wachstein and Besen, 1964) i n enzymatic a c t i v i t y of the kidney. Padykula and Herman (1955b) examined kidneys of rats, while Freiman and Kaplan (1959) used those of dogs. In a l l pro b a b i l i t y , both a l k a l i n e phosphatase and ATPase are present i n the brush border of the rat kidneys studied here. The precise function of the ATPase associated with the brush border i s not e n t i r e l y c l e a r but i t i s most l i k e l y involved i n the transport of substances from the tubular lumens into the c e l l s and vice versa i n the process of urine formation from the glomerular f i l t r a t e . It i s an energy-requiring process inv o l v i n g the movement of substances up an electrochemical p o t e n t i a l gradient. This, by d e f i n i t i o n , i s "active trans- port" (Solomon, 1962). Wot only ATPase (Spater et a l . , 1958; • Wheeler and Whittam, 1964; Ericsson and Trump, 1969) but also a l k a l i n e phosphatase (Wilmer, 1944; Rosenberg and Wil- brandt, 1952; Kissane, 1961; Matthiessen, 1966) have been implicated to take part i n active transport across membranes. In much of the l i t e r a t u r e on the histochemical l o c a l i s a t i o n of ATPase i n various tissues, there i s a tendency to consider the ATPase so l o c a l i s e d as associated only with the sodium pump (Solomon, 1962; Post and Sen, 1965). This ATPase i s sodium-potassium activated and ouabain-sensitive. There i s much controversy as to whether the sodium-potassium a c t i v - 38. ated ATPase i s demonstrable at a l l with the present avai l a b l e • histochemical techniques (Bonting et a l . , 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, esp e c i a l l y with respect to the k i d - ney. Besides p a r t i c i p a t i n g i n the active transport of cations l i k e sodium and potassium, i t perhaps also participates i n the active transport of anions l i k e phosphate and sulfate, sugars, amino-acids and f a t t y acids (loewy and Siekevitz, 1963). It i s not known whether there i s a common mechanism underlying the active transport of a l l these substances and therefore involv-' ing one common ATPase, or whether a number of ATPases are work- ing i n concert. The l a t t e r suggestion seems more plausible. In the brush border of the proximal tubules, small molecules presumably enter the c e l l s by active transport across the plasma membranes. Larger molecules, c o l l o i d a l mat- e r i a l s and proteins probably enter by way of pinocytosis. The substances which are to be absorbed stream towards the bases of the m i c r o v i l l i where v e s i c l e s and vacuoles of various sizes are pinched o f f . This form of membrane flow probably requires energy (Loewy and Siekevitz, 1963) and therefore an ATP- dephosphorylating enzyme to make the energy a v a i l a b l e . Ericsson (1965a, 1965b) i n h i s studies of the transport and digestion of hemoglobin, found that the hemoglobin was r a p i d l y pino- cytosed and metabolised. Droplets containing hemoglobin app- eared a l l below the brush border. Four hours a f t e r the i n t r a - venous i n j e c t i o n of hemoglobin, the brush border of the prox- imal tubules was abolished. Ericsson postulated that the brush' border membrane was being used up f o r absorption droplets and that membrane renewal was not keeping pace with membrane lo s s . Under ph y s i o l o g i c a l conditions, membrane renewal and loss would be balanced. Renewal of the plasma membrane of the brush border i s conceivably an energy-requiring process with an associated ATPase. The b a s i l a r and l a t e r a l i n t e r d i g i t a t i n g membranes tremendously increase the surface area of the c e l l i n contact 39. with the e x t r a c e l l u l a r space. There i s a large number of mito- chondria cl o s e l y apposed to these membranes where transport processes can occur. These membranes show an intense ATPase reaction. It has been observed that the e x t r a c e l l u l a r spaces widen i n response to conditions of f i x a t i o n and also to var- ious solutions injected intravenously ( C a u l f i e l d and Trump, 1962) by increased water fluxes. The ATPase i n the b a s i l a r regions of the proximal tubule c e l l s presumably transport large amounts of s a l t s and water. Where l i t t l e c o r r e l a t i o n between structure, function and enzymatic a c t i v i t y i n the proximal tubules i s possible, there i s an even lesser p o s s i b i l i t y i n the d i s t a l tubules. The ATPase i n the d i s t a l 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 t h i s i s not known. The functions post- ulated f o r the d i s t a l tubules include active transport of sodium, potassium secretion, a c i d i f i c a t i o n of the urine and ammonia secretion (Ericsson and Trump, 1969). A precise c o r r e l a t i o n i s not possible at the present time. In the glomerulus, enzymatic a c t i v i t y was l o c a l i s e d 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 a l . , 1963; Wachstein and Besen, 1964). The ATPase i n the glomerulus responded to both PHMB and L-cysteine. This could just be an ind i c a t i o n of the glomerulus' s e n s i t i v i t y to any foreign' substances. It was once thought that the foot processes had only a supportive function i n the glomerulus and that substances once past the tril a m i n a r basement membrane, flowed i n between the f i l t r a t i o n s l i t s set up by the i n t e r d i g i t a t i n g foot processes. There i s now a suggestion that transport processes do occur across the glomerular e p i t h e l i a l membranes espe c i a l l y i n r e l a t i o n to resorption of proteins that may have f i l t e r e d across the endothelial pores and basement membrane (Jones, 1969). 40. The author postulates that i n the adult rat kidney, at least two types of ATPases are l o c a l i s e d 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 i n response to the modifiers PHMB and L-cysteine. The enzyme with an optimum at pH 9.4 appears to be more sens- i t i v e to the e f f e c t s of modifiers. Unlike the four types of ATPases i n the muscle which can be s p a t i a l l y separated i n terms of t h e i r pH optima and t h e i r response to various activators and i n h i b i t o r s (Gauthier, 1967), those i n the kidney are both l o c a l i s e d at the plasma membranes. This postulate, of course, i s highly speculative. (B) Newborn kidney In the r a t , the newborn kidney i s not only morpho- l o g i c a l l y but also f u n c t i o n a l l y immature, fas compared to the adult. The newborn kidney i s an i n e f f i c i e n t homeostatic organ and. cannot tolerate, changes i n acid and base intake (Wacker et a l . , 1961; Moog, 1965). The newborn rats cannot handle changes' i n hydration (Pinkstaff et a l . , 1962) which i s e s p e c i a l l y evident i f they are overloaded with water (Wachstein and'Brad- shaw, 1965). Rates of glomerular f i l t r a t i o n , urea clearance, absorption of glucose and accumulation of v i t a l dyes are low when compared with those of the adult (Wachstein and Bradshaw, 1965). This f u n c t i o n a l i n e f f i c i e n c y can generally be correlated with a le s s than f u l l complement of enzymes. With morphological development, the enzymes f o r p h y s i o l o g i c a l function appear and/or increase i n amount. Enzyme accumulation can be taken as one aspect of f u n c t i o n a l d i f f e r e n t i a t i o n (Pinkstaff et a l . , 1962). That there i s morphological immaturity i n the newVx- • born kidney i s 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 f o r the transport of substances from the glom- erular f i l t r a t e i s small. There i s no brush border, or i f 41. present, only i n a r e l a t i v e l y undeveloped form. The i n t e r - d i g i t a t i o n s of the basal and l a t e r a l membranes are scarce. The glomeruli too, are mostly morphologically undifferentiated. The endothelium possess few fenestrations and the glomerular e p i t h e l i a l c e l l s have few foot processes. Concomitant with the observations of morphological immaturity i s the observat- ion that the enzyme ATPase i s r e l a t i v e l y l e s s abundant. In the tubules, enzymatic activirfey was noted mainly on the l a t e r a l membranes i n between i n d i v i d u a l c e l l s . There was no great accumulation of reaction p r e c i p i t a t e on the basal membranes or luminal surface plasma membranes, undifferentiated morpho- l o g i c a l l y though they may be, as i n the adult. Only with the d i f f e r e n t i a t i o n of these membranes, as i n a developing prox- imal tubule, i s there accumulation of reaction product, and therefore an i n d i c a t i o n of increased enzymatic a c t i v i t y . The weak reaction f o r ATPase enzymatic a c t i v i t y on the luminal surface membrane and few m i c r o v i l l i of the undifferentiated tubules would indicate some transport of substances from the tubular lumens into the c e l l s . The shortest possible route f o r substances out into the e x t r a c e l l u l a r space would be v i a the l a t e r a l membranes, where thereis intense reaction f o r ATPase enzymatic a c t i v i t y . These observations do not agree with those of Wachstein and Bradshaw (1965) who found no tubular ATPase i n the newborn kidney, even i n the more mature_developin_r_>prox- imal tubules. However, Pinkstaff et a l . (1962) showed ATPase a c t i v i t y i n a l l f e t a l and post-natal stages. The endothelium i n the immature glomerulus often showed deposition of reaction product. Possibly active transport occurs across the endo- thelium of the newborn glomerulus to compensate f o r th© pauc- i t y of fenestrae through which glomerular f i l t r a t e can pass. The glomerular e p i t h e l i a l c e l l s showed much reaction precip- i t a t e associated with the plasma membranes which were i n apposition with other membranes, thus probably delineating the pathway f o r glomerular f i l t r a t e from the c a p i l l a r i e s to Bowman's space. As with the adult, i t i s postulated that there are 42. probably at least two types of enzymes present i n the new- born kidney with pH optima at 7.2 and 9.4. At b i r t h , the ATP- ase active at pH 7.2 i s probably abundant and stable and there- fore more resist a n t to f i x a t i o n e f f e c t s . At pH 9.4, the eff e c t s of f i x a t i o n on enzymatic a c t i v i t y of the newborn kidney were very apparent. We can only speculate as to why t h i s i s so. This enzyme with a pH optimum at 9.4 could be only present i n small amounts, or was unstable, or was i n an inactivated form, or a combination of a l l these three p o s s i b i l i t i e s . With a diminished t o t a l plasma membrane surface area, i t i s conceivable to have a diminished amount of enzymes associated with i t . During de- velopment enzymes may change from the inactive form to the active form as well as change i n s t a b i l i t y (Moog, 1965). The increase i n the quantity of enzymes demonstrable histochemic- a l l y could re s u l t from protein synthesis ( P r i e s t l y and Malt, 1968) or an ac t i v a t i o n of enzymes previously i n an inactive form. c - SUMMARY OF ENZYMATIC AVTIVITY OF THE ADULT AND NEWBORN KIDNEY The f u n c t i o n a l capacity of both the adult and new- born kidney can be correlated with the extent of i t s morpho- l o g i c a l d i f f e r e n t i a t i o n and with the amounts of enzymes present. The adult kidney maintains body homeostasis e f f i c i e n t - l y . The tubular elements i n the cortex and the glomeruli are u l t r a s t r u c t u r a l l y complex. The amount of ATPase enzymatic a c t i v i t y demonstrable histochemically i s considerable. The enzymes appear to be highly stable and are not affected by long periods of pre- f i x a t i o n i n glutaraldehyde p r i o r to incubation i n the ATP substrate medium. In the proximal tubules, the reaction pr e c i p i t a t e i s l o c a l i s e d on the memb- ranes of the brush border m i c r o v i l l i , t£e l i m i t i n g membranes of some of the a p i c a l v e s i c l e s and vacuoles, and the membranes of the basal i n t e r d i g i t a t i o n s . The brush border ATPase i s probably related to the active transport across plasma membranes of small molecules from the tubular lumens into the 43. c e l l s and vi c e versa (Loewy and Siekevitz, 1963). Larger mol- ecules, c o l l o i d a l materials and proteins perhaps enter the c e l l s by pinocytosis. Some of the tubular invaginations from the bases of the m i c r o v i l l i and some of the a p i c a l v e s i c l e s and vacuoles sometimes show enzymatic a c t i v i t y . These c e l l u l a r components associated with pinocytosis might represent a form of membrane flow within the c e l l which i s energy-requiring (Loewy and Siekevitz, 1963). The basal infoldings divide the cytoplasm into numerous compartments within which are contained large, elongated mitochondria. These basal i n t e r d i g i t a t i o n s are highly sensitive to conditions of f i x a t i o n and to sol u t - ions injected intravenously ( C a u l f i e l d and Trump, 1962). It i s suggested that the ATPases i n the b a s i l a r parts of the proximal tubule c e l l s are associated with the transport of s a l t s and water. In the d i s t a l tubules, i t i s d i f f i c u l t to correlate the ATPase enzymatic a c t i v i t y present on the b a s i l a r i n f o l d i n g membranes with some of the postulated functions of the d i s t a l tubules, f o r example,.transport of sodium, secret- ion of potassium and ammonia, and a c i d i f i c a t i o n of the urine (Ericsson and Trump, 1969). In the glomerulus, reaction p r e c i p i t a t e i s observed on the podocytic foot processes. Besides having a mechanical supportive function, t^re podocytic c e l l s probably a c t i v e l y resorb proteins that have f i l t e r e d through the endothelial fenestrae and basement membrane (Jones, 1969). The newborn kidney, as compared with the adult kidney, i s f u n c t i o n a l l y immature, morphologically undifferent- iated and enzymatically l e s s adequately endowed (Wacker et a l . , 1961; Pinkstaff et a l . , 1962; Moog, 1965; Wachstein and Brad- shaw, 1965). Enzymatic a c t i v i t y that i s demonstrable h i s t o - chemically i s l o c a l i s e d on the l a t e r a l membranes between i n d i v i d u a l c e l l s of the undifferentiated tubules, on the apposed membranes of podocytic c e l l s and sometimes i n the endothelium. The presence of these enzymes would show that the newborn kidney, though s t i l l immature, i s capable of carrying out many of the functions e s s e n t i a l f o r maintaining homeo- 44. s t a s i s . In the undifferentiated tubules there i s no brush border with i t s complement of enzymes. This would perhaps ex- p l a i n , p a r t i a l l y , the low rate of glucose absorption i n the newborn kidney (Wachstein and Bradshaw, 1965). Newborn rats cannot eliminate excess water from t h e i r bodies as e f f i c i e n t - l y as the adults (Pinkstaff et a l . , 1962; Wachstein and Brad- shaw, 1965). I f water i s mainly transported across the memb- ranes of the b a s i l a r i n t e r d i g i t a t i o n s of the proximal tubules c e l l s , then the lack or paucity of b a s i l a r infoldings with the corresponding absence of enzymatic a c t i v i t y , would account f o r the decreased functional capacity i n terms of water movement. For both the adult and the newborn kidneys, i t i s postulated that there are at least two types of ATPases - l o c a l i s e d at the plasma membranes with pH optima of 7.2 and 9.4. In the adult, the enzymes appear to be unaffected by pre- f i x a t i o n i n glutaraldehyde. In the newborn however, the enzyme active at pH 9.4 seems to be sensitive to the length of pre- f i x a t i o n . D. NUCLEAR STAINING The significance of nuclear staining i n tissues incubated f o r the ATPase enzymatic reaction i s s t i l l i n abey- ance. Nuclear staining i s frequently observed (Padykula and Herman, 1955a; Novikoff et a l . , 1958; Holt, 1959; Pinkstaff et a l . , 1962; Sandler and Bourne, 1962; Wachstein and Brad- shaw, 1962; Ashworth et a l . , 1963; Deane, 1963; Tewari and Bourne, 1963a, 1963b; McClurkin, 1964; Wachstein and Besen, 1964; Kl e i n , 1966; Moses et a l . , 1966; Tasuzumi and Tsubo, 1966; Jacobsen and Jorgensen, 1969). In most instances, as with the author's observations, nuclear staining i s e r r a t i c and inconsistent and i s considered as a r t i f a c t u a l rather than as a manifestation of enzymatic a c t i v i t y . The mechanism of nuclear staining i s not clear but the most commonly given reason i s an a f f i n i t y of n u c l e i f o r lead and calcium phosph- ates (Ashworth et a l . , 1963; Deane, 1963; Moses et a l . , 1966). In some instances, investigators have found a consistent 45. pattern of nuclear staining. Pinkstaff et a l . (1962) observed nuclear staining i n the proximal tubules i n a l l f e t a l and post-natal stages, and an increasing nuclear staining i n d i s t - a l tubules. Sandler and Bourne (1962) could produce or cause to vanish nuclear s t a i n i n g by varying the magnesium sulfate concentration i n the incubating media. Others have found nuc- l e a r staining to be dependent on the concentration of ATP and of lead i n the medium (Padykula and Herman, 1955a; Novikoff et a l . , 1958; Moses et a l . , 1966). Yasuzumi and Tsubo (1966) by modifying the histochemical method used cou-ld l o c a l i s e react- ion precipitate i n the region of the nuclear pores. Some spec- ulations as to the function of t h i s nuclear ATPase have been put forward. This ATPase, i f present, would hint at a meta- b o l i c interaction between the n u c l e i and the cytoplasm (Sand- l e r and Bourne, 1962; Tewari and Bourne, 1963a, 1963b; McClurkin, 1964; Yasuzumi and Tsubo, 1966). E. CONTROLS In p r a c t i c a l l y a l l control specimens, no pr e c i p i t a t e was observed. But occasionally there was p r e c i p i t a t e assoc- iated with the brush border of the proximal tubules (Persijn et a l . , 1961; Wachstein and Besen, 1964) when the method of Wachstein and Meisel (1957) was used. The pr e c i p i t a t e i s elec- tron-dense but the nature of the composition of the p r e c i p i t - ate i s not at a l l c l e a r . In the present study, i t was observed that there was a tendency f o r accumulation of p r e c i p i t a t e to occur as the m i c r o v i l l i developed i n length and i n complexity. Perhaps with d i f f e r e n t i a t i o n , the m i c r o v i l l i membranes acquire the glycocalyx coat which has an a f f i n i t y f o r the p r e c i p i t a t e s formed as a result of the in t e r a c t i o n between the various components of the incubating media. There does not appear to be t h i s s e l e c t i v e a f f i n i t y f o r precipitates on the glomerular podocytic c e l l membranes where a coat of mucosubstances i s also present (Jones, 1969). 46. ILLUSTRATIOHS Figures 1 - 4 Enzymatic a c t i v i t y of the adult kidney at pH 7.2 5 - 10 Enzymatic a c t i v i t y of the newborn kidney at pH 7.2 11 - 17 Enzymatic a c t i v i t y of the newborn kidney at pH 9.4 1 8 - 2 5 Effect of modifiers on enzymatic a c t i v i t y of the adult kidney 26 - 36 E f f e c t of modifiers on enzymatic a c t i v i t y 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 c a p i l l a r y N nucleus M mitochondrion E p a r i e t a l epithelium Et erythrocyte p podocyte f podocytic foot process a small a p i c a l v e s i c l e v large a p i c a l vacuole t tubular invagination m mi c r o v i l l u s b brush border e endothelium bm basement membrane 47. 7 >V.> _ ' V - , _ ^ ; v a n a a a * , • . * • % « -—a M Figure 1 5 hours pr e - f i x a t i o n i n glutaraldehyde. A proximal tubule. The brush border (b) of the proximal tubule from an adult kidney i s exten- sive. In response to the mode of f i x a t i o n , the m i c r o v i l l i are closely packed together and the tubular lumen i s ob l i t e r a t e d . In the a p i c a l cytoplasm are a number of tubular invaginations ( t ) , small a p i c a l v e s i c l e s (a) and larger a p i c a l 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 i s present i n the brush border, i n the cytoplasm and on the cytoplasmic aspect of the i n f o l d i n g membranes. x 11,800 48 Figure 2 5 hours pre-fixation i n glutaraldehyde. A proximal tubule. Some of the i n t e r d i g i t a t - ions of the basal membranes are c l e a r l y shown. The e x t r a c e l l u l a r compartments (arrows) formed by the i n f o l d i n g membranes are s l i g h t l y en- larged i n response to the ef f e c t s of f i x a t i o n . The reaction p r e c i p i t a t e , i n the form of clumps, i s found adhering to the i n t e r d i g i t a t i n g memb- ranes and i s also present i n the basement membrane (bm). x 34,300 49. Figure 3 5 hours pre-fixation i n glutaraldehyde. A d i s t a l tubule. There i s a much more e l - aborate system of i n t e r d i g i t a t i o n s of the basal membranes.A large number of elongated mitochondria (M) are cl o s e l y associated with the membranes. The reaction precipitate i s present on the cytoplasmic aspects of the membranes as well as within the cytoplasm. x 16,390 50. Figure 4 5 hours pre-fixation i n glutaraldehyde. A glomerulus. The basement membrane (bm) separating the fenestrated endothelium (e) from the i n t r i c a t e pattern of podocytic foot processes (f) i s prominent. One podocytic c e l l (p) may send out cytoplasmic processes to more than one c a p i l l a r y (arrows).Fine reaction pre- c i p i t a t e i s seen on the membranes and i n the cytoplasm of the podocytic foot processes, i n the basement membrane but not i n the endothelium. x 11,800 51 Figure 5 2 hour-old kidney. 2 hours pre-fixation i n glutaraldehyde. An undifferentiated tubule. There i s intense enzymatic reaction on the l a t e r a l membranes between the i n d i v i d u a l c e l l s . The plasma memb- rane l i n i n g the luiften (L) shows no sign of enzymatic a c t i v i t y . Only one of the tubular c e l l s (arrow) has reaction p r e c i p i t a t e assoc- iated with the simple basal membrane. x 4,700 52. Figure 6 2 hour-old kidney. 2 hours pre-fixation i n glut arald ehyd e. An undifferentiated tubule. Reaction p r e c i p i - tate i s abundant on the l a t e r a l membranes. At the luminal surface (L) a small amount of preci p i t a t e i s present on the plasma membrane of the few short m i c r o v i l l i (m) as well as witfrin the core of the m i c r o v i l l i . There i s no reaction on the basal membranes. x 9,100 53. F i g u r e 7 2 hour-old kidney. 2 hours pre-fixation i n glut arald ehyd e. An undifferentiated tubule. The deposition of the reaction product f o r ATPase a c t i v i t y on the l a t e r a l membranes (arrows) c l e a r l y demar- cates the l a t e r a l boundaries of each c e l l i n the tubule. The basal membranes show some enzymatic a c t i v i t y . However, there i s no pre- c i p i t a t e on the luminal (L) surface plasma membrane. x 9,100 54. Figure 8 24 hour-old kidney. 5 hours p r e - f i x a t i o n i n glutaraldehyde. A developing proximal t u b u l e . U n l i k e the un- d i f f e r e n t i a t e d tubule, the brush border (b) i s r e l a t i v e l y well-developed and there are a l a r g e number of sm a l l a p i c a l v e s i c l e s ( a ) , l a r g e a p i c a l vacuoles (v) and mitochondria (M). In the lumen (L) there i s a probably degenerating c e l l . The f i n e r e a c t i o n p r e c i p i t a t e encrusts the micro- v i l l i of the brush border ( b ) . There i s some r e a c t i o n product on the membranes of the dev- e l o p i n g b a s a l i n t e r d i g i t a t i o n s (arrows). x 4,700 55. F i g u r e 9 2 hour-old kidney. 2 hours pre - f i x a t i o n i n glut arald ehyd e. An immature glomerulus. The podocytic c e l l s are closely packed together. Reaction i s most prominently observed where two sets of memb- ranes are i n apposition (arrows). x 9,100 56. Figure 10 24 hour-old kidney. 5 nours pre-fixation i n glut araId ehyd e. A s l i g h t l y more d i f f e r e n t i a t e d glomerulus than i n Figure 9 . The podocytic c e l l body (p) i s s t i l l situated close to the c a p i l l a r y (C). There are a number of podocytic foot processes (f) abutting on the trilaminar basement membrane (bm). The endothelial c e l l (e) i s becoming fenestrated (arrows). The reaction p r e c i p i t a t e i s found not only on the c e l l membranes but also within the cytoplasm of the podocytic c e l l body, the podocytic foot processes and endothelium. There i s also quite a b i t of prec i p i t a t e assoc- iated with the denser heterochromatin regions of the podocytic c e l l nucleus (N). x 11,800 57. F i g u r e 11 2 hour-old kidney. 2 hours pre-fixation i n glut arald ehyd e. An undifferentiated tubule. Morphologically the tubular c e l l s are well preserved except f o r a few "exploded" mitochondria (M). With 2 hours pre-fixation, there i s almost always no demonstrable enzymatic reaction. Occasion- a l l y a few tubular c e l l s show discrete part- i c l e s of reaction p r e c i p i t a t e on the plasma membrane l i n i n g the lumen (L). There i s no reaction on the l a t e r a l membranes (arrows) or basal membranes. x 9,100 58. Figure 12 1 hour-old kidney. 1 hour pre - f i x a t i o n i n glut arald ehyd e. An undifferentiated tubule. As i n Figure 11 there i s only a small amount of prec i p i t a t e associated with the membrane l i n i n g the tub- u l a r lumen (L). There i s no enzymatic a c t i v i t y on the l a t e r a l membranes (arrows) or basal membranes. x 11,800 59. Figure 13 1 hour-old kidney. 15 mins. pre-fixation i n glutaraldehyde. A developing proximal tubule. The m i c r o v i l l i (m), though s t i l l few i n number, are becoming long and slender. U l t r a s t r u c t u r a l l y the c e l l s do not appear to be so well preserved as i n Figures 11-12. However, with a shortened pre- f i x a t i o n period i n glutaraldehyde (15 mins. instead of 1 hour) enzymatic a c t i v i t y i s pre- sent on the m i c r o v i l l i membranes (m) and on the l a t e r a l membranes (arrows). There i s some preci p i t a t e i n the region of the basement membrane (bm). x 7,500 60. Figure 14 12 hour-old kidney. 2 hours pre-fixation i n glut araId ehyd e. A developing proximal tubule. The m i c r o v i l l i (m) of the r e l a t i v e l y well-developed brush border are coated with reaction p r e c i p i t a t e deposits. The tubular invaginations (t) from the bases of the m i c r o v i l l i (m), the small a p i c a l v e s i c l e s (a) and some of the large ap- i c a l vacuoles (v) also show the presence of reaction product. There i s an intense reaction on the l a t e r a l membranes (arrows). At the base of the tubular c e l l s , some prec i p i t a t e i s pre- sent but i t i s not clea r whether the p r e c i p i - tate i s associated with the basal membranes or the basement membrane. x 9,100 61. Figure 15 3 day-old kidney. 5 hours pre-fixation i n glut arald ehyd e. Brush border of a developing proximal tubule. The brush border (b) i s quite extensive. It i s in a collapsed state as a resu l t of the imm- ersion method of f i x a t i o n used. It i s c l e a r l y seen that the f i n e precipitate encrusts the membranes of the m i c r o v i l l i . A number of the tubular invaginations from the bases of the micro- v i l l i (arrows) show a marked deposition of the reaction p r e c i p i t a t e . The few small a p i c a l vac- uoles (v) that are present have no reaction p r e c i p i t a t e . x 11,800 Figure 16 12 hour-old kidney. 2 hours pr e - f i x a t i o n i n glut araId ehyd e. An immature glomerulus. This group of six podocytic c e l l s show var i a t i o n s i n response to incubation i n the ATP substrate medium. Some c e l l s show a weak reaction ( l , 2) while others a more pronounced reaction (3, 4, 5). The podo- cyte i n the centre (6) i s not reactive except where i t s c e l l membrane i s i n apposition with the c e l l membrane of another podocyte (5) (arrow). x 7,500 63. Figure 17 12 hour-old kidney. 2 hours pre-fixation i n glut araId ehyd e. An immature glomerulus. The podocytic c e l l s (p) are cl o s e l y packed together at the basal poles but the a p i c a l poles are free i n Bowman's space (L). There are no foot processes. The endothelium (e) has not assumed i t s fenestrated form. It i s very apparent that there i s an intense ATPase reaction where the membranes of the podocytic c e l l s are i n apposition with the membranes of the neighbouring c e l l s (arrows), but not i f they are free and exposed i n Bow- man' s space (L). The endothelial c e l l s (e) have n o reaction p r e c i p i t a t e . x 4,700 64. Figure 18 3 hours pr e - f i x a t i o n i n 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 i n enzymatic a c t i v i t y . In one tubule there are two d i s t i n c t regions of enzymatic a c t i v i t y ; at the brush border (b) and basal i n t e r d i g i t - ations (arrows). The basal infoldings of the other tubule show no deposition of reaction pr e c i p i t a t e . x 7,500 65. Figure 19 3 hours pre-fixation i n glutaraldehyde. No pre- incubation exposure to PHMB. Incubation with PHMB at pH 7.2. Brush border of a proximal tubule. There i s some ind i c a t i o n of an electron-dense material within the core of the m i c r o v i l l i ( c i r c l e ) . The reaction precipitate i s not uniformly d i s t - ributed over the membranes of a l l the m i c r o v i l l i i n the brush border. Instead clumps of p r e c i p i t - ate are observed i n various regions of the brush border (arrows). This i s probably a r t i f a c t u a l rather than a result of heterogeneity of response to PHMB. x 11,800 66. Figure 20 3 hours pre-fixation i n glutaraldenyde. No pre- incubation exposure to PHMB. Incubation with Pfflffl at pH 7.2. Glomerulus. This i s a low magnification electron micrograph of a glomerulus showing various cap- i l l a r i e s (C) and the i n t r i c a t e network of podo- c y t i c foot processes (f) on the tri l a m i n a r base- ment membrane separating the endothelium (e) from the v i s c e r a l epithelium. The reaction pre- c i p i t a t e i s dis t r i b u t e d a l l along the plasma membranes of the podocytic foot processes but not on the membranes of the endothelial c e l l s . x 3,700 67. Figure 21 3 hours pre-fixation i n glutaraldehyde. Pre- incubation exposure to PHMB. Incubation with PHMB at pH 7.2. Glomerulus. The fenestrated endothelium (arrows) of the c a p i l l a r i e s (C) i s separated from the podocytic foot processes (f) by a d i s t i n c t l y v i s i b l e basement membrane (bm). There i s no reaction p r e c i p i t a t e on the endothelium. How- ever reaction pr e c i p i t a t e i s present a l l along the plasma membranes of the podocytic foot processes. x 9,100 68. Figure ZZ 3 hours pre-fixation i n glutaraldehyde. Pre- incubation exposure to PHMB. Incubation with PHMB at pH 9 . 4 . Proximal tubule. The basal i n t e r d i g i t a t i o n s i n the proximal tubule are quite extensive. Some may be confined to the basal portions of the c e l l while others may extend almost to the bases of the brush border (b). The e x t r a c e l l u l a r compart- ments formed by the i n t e r d i g i t a t i o n s are very sensitive to the effects of f i x a t i o n and are often separated, as exemplified here. There i s no ATP- ase a c t i v i t y on these membranes although enzy- matic a c t i v i t y i s re a d i l y observed i n the brush border (b) and i n the tubular invaginations (arrows) a r i s i n g from the bases of the m i c r o v i l l i . Only one a p i c a l vacuole (v) has some reaction pr e c i p i t a t e associated with i t . x 5,700 69. Figure 25 3 hours pre - f i x a t i o n i n glutaraldehyde. No pre- incubation exposure to PHMB. Incubation with PHMB at pH 9.4. Proximal tubule. The enlarged e x t r a c e l l u l a r compartments (arrows) bound by the i n f o l d i n g basal membranes indicate the s e n s i t i v i t y of the basal regions of the proximal tubules to the effects of f i x a t i o n . Not a l l the e x t r a c e l l u l a r compartments are enlarged to the same extent. Unlike Figure 22, the basal i n t e r d i g i t a t i o n s show an intense enzymatic reaction. The fin e reaction p r e c i p i t a t e i s seen adhering to the membranes and i s not free i n the e x t r a c e l l u l a r space. The basement membrane (bm) i s r e l a t i v e l y thick and i s generally free of any pre c i p i t a t e . x 11,800 70. Figure 24 3 hours pre-fixation i n glutaraldehyde. Pre- incubation exposure to PHMB. Incubation with PHMB at pH 9.4. Brush border of a proximal tubule. There i s an abundant deposition of reaction precipitate along the membranes of the m i c r o v i l l i making up the brush border. A large number of the tubular invaginations from the bases of the m i c r o v i l l i (arrows) are also coated with the reaction pre- c i p i t a t e . Some small a p i c a l v e s i c l e s (a) have reaction product on t h e i r l i m i t i n g membranes. The large a p i c a l vacuoles (v) that are present have no accumulation of reaction p r e c i p i t a t e . x 11,800 Figure 25 3 hours pr e - f i x a t i o n i n glutaraldehyde. No pre- incubation exposure to PHMB. Incubation with PHMB at pH 9.4. Glomerulus. The fi n e reaction p r e c i p i t a t e i s observed not only along the membranes of the podocytic foot processes (f) but also within the cytoplasm (compare with Figures 20, 21). There i s some precipitate i n the basement memb- rane (bm) which i s probably due to d i f f u s i o n of reaction precipitate from the adjacent podo- c y t i c foot processes. The fenestrated endo- thelium (e) shows no enzymatic a c t i v i t y . x 16,300 Figure 26 2 hour-old kidney. 2 hours pre-fixation i n gluta raldehyde. No pre-incubation exposure to PHMB. Incubation with PHMB at pH 7.2. An undifferentiated tubule. The plasma membrane l i n i n g the lumen (L) as well as the basal memb- ranes (arrows) of the tubular c e l l s are s t i l l simple i n contour. The l a t e r a l membranes show the beginnings of i n t e r d i g i t a t i o n s which w i l l become much more complex with continuing d i f f - erentiation. Heavy deposits of reaction pre- c i p i t a t e on the l a t e r a l membranes accentuate the l a t e r a l boundaries between the tubular c e l l s There i s no reaction p r e c i p i t a t e on the basal membranes or on the luminal surface membrane. x 4,700 73. Figure 27 2 hour-old kidney. 2 hours pr e - f i x a t i o n i n 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 i s s t i l l r e l a t i v e l y smooth although a few m i c r o v i l l i are present. The basal membranes of some c e l l s are beginning to i n f o l d (arrows). The l a t e r a l membranes also show some i n t e r d i g i t a t i o n s . There i s intense enzymatic reaction a l l along the luminal surface plasma membrane, the i n t e r d i g i t a t i n g l a t e r a l membranes and the membranes of the basal i n f o l d - ings, wherever they are present. A few of the nuc l e i (N) show an accumulation of fi n e p r e c i p i t a t e . x 5,700 74. Figure 28 2 hour-old kidney. 2 hours pr e - f i x a t i o n i n gluta- raldehyde. Pre-incubation exposure to PHMB. Incubation with PHMB at pH 7.2. An undifferentiated tubule. U l t r a s t r u c t u r a l l y the tubular c e l l s are simple. Each c e l l consists of a large nucleus (N) and a few mitochondria (M) contained within a small amount of cytoplasm. At the at»ex of the c e l l there are a few micro- v i l l i (m). Part of the a p i c a l cytoplasm appears to be blebbing o f f (arrows) and discarded into the lumen (L) as c e l l u l a r debris. The l a t e r a l membranes follow a straight path from the lumen to the base of the c e l l . The basal membranes show no complex i n t e r d i g i t a t i o n s as i n the adult. There i s p r a c t i c a l l y no demonstrable enzymatic a c t i v i t y except f o r part of the l a t e r a l membrane, ( c i r c l e ) . x 7,500 75. Figure 29 2 hour-old kidney. 2 hours pre-fixation i n gluta- raldehyde. No pre-incubation exposure to PHMB. Incubation with PHMB at pH 7.2. An undifferentiated tubule. A l l , but one, of the c e l l s i s normal and show deposition of re- action p r e c i p i t a t e on the l a t e r a l membranes. This one c e l l i s "exploded." The contents of the c e l l , that i s , the cytoplasm and mitochondria (M) are being s p i l l e d out into the lumen (L). The nucleus (N) i s swollen to immense proportions. The nuc- lea r membrane (arrows) appears i n t a c t . The chrom- a t i n material adheres to the nuclear membrane or i s suspended i n the nucleoplasm. The reason f o r t h i s p a r t i c u l a r c e l l 1 s extreme s e n s i t i v i t y to experimental conditions i s not known. x 7,500 76. •Figure 50 2 hour-old kidney. 2 hours pre-fixation i n gluta- raldehyde. No pre-incubation exposure to PHMB. Incubation with PHMB at pH 7.2. A developing proximal tubule. The tubule shows the f i r s t d e f i n i t e signs of d i f f e r e n t i a t i o n into a proximal tubule. The m i c r o v i l l i (m) are s t i l l sparse but r e l a t i v e l y long and slender. There are a number of a p i c a l v e s i c l e s (a) i n the a p i c a l cytoplasm. The l a t e r a l and basal membranes are simple i n contour. Reaction precipitate i s deposited heavily on the l a t e r a l membranes (arrows), the luminal (L) surface membrane and the membranes of the m i c r o v i l l i . Some of the small a p i c a l v e s i c l e s (a) have reaction p r e c i p i t a t e a l l along the l i m i t - ing membranes. x 7,500 77. Figure 51 2 hour-old kidney. 2 hours pr e - f i x a t i o n i n gluta- raldehyde. Pre-incubation exposure to PHI-IB. Incubation with PHMB at pH 7.2. A developing proximal tubule. This i s a tubule further along i n d i f f e r e n t i a t i o n (compare with Figure 30). The brush border (b) i s quite well- developed, the l a t e r a l membranes pursue a s l i g h t l y more tortuous course from the lumens to the base of the c e l l (arrows) and some i n t e r d i g i t a t i o n s of the basal membranes are seen ( c i r c l e ) . There are a large number of tubular invaginations (t) from the bases of the m i c r o v i l l i , small a p i c a l v e s i c l e s (a) and large a p i c a l vacuoles (v). Some of the small, round mitochondria (M) that are present appear to be sensitive to the effects of f i x a t i o n . Enzymatic a c t i v i t y i s most prominent i n the brush border (b), the tubular invaginations ( t ) , and on the l a t e r a l and basal i n t e r d i g i t a t i o n s . x 3,700 78. Figure 52 2 hour-old kidney. 2 hours pre - f i x a t i o n i n gluta raldehyde. Pre-incubation exposure to PHI-IB. Incubation with PHMB at pH 7.2. A developing proximal tubule. This tubule i s more d i f f e r e n t i a t e d than those i n Figures 30 and 51. The brush border (b) and the basal i n t e r d i g i t a t i o n s are quite extensive. Cytoplasmic compartments with associated mitochondria (M) are developing. Most of the mitochondria are s t i l l small and round (Ml, M2) while others are becoming elongate (M5, M4). The reaction precipitate i s observed on the membranes of the basal i n t e r d i g i t a t i o n s , the brush border, tub- u l a r invaginations (t) and small a p i c a l v e s i c l e s Ca). x 9,100 79. e Figure 55 2 hour-old kidney. 2 hours pre-fixation i n gluta- raldehyde. Fo pre-incubation exposure to PHMB. Incubation with PHMB at pH 7.2. An immature glomerulus. The p a r i e t a l epithelium (E-)- delineates the outermost extent of the glom- erulus. The v i s c e r a l epithelium, made up of the podocytic c e l l bodies (p) and the beginnings of foot processes ( f ) , surround the endothelium (e) of two c a p i l l a r i e s . The c a p i l l a r y lumens are small but can be recognised by the presence of an erythrocyte ( E t ) . The a p i c a l poles of the podo- c y t i c c e l l s are free i n Bowman's space (L) while the foot processes from the basal poles abutt on the basement membrane around the c a p i l l a r i e s . Enzymatic a c t i v i t y i s only present on the memb- ranes of the foot processes i n di r e c t contact with the basement membrane, and also on the l a t e r a l membranes of the p a r i e t a l e p i t h e l i a l c e l l s . x 3,700 80. Figure 54 2 hour-old kidney. 2 hours pre-fixation i n gluta raldehyde. No pre-incubation exposure to L- cysteine. Incubation with L-cysteine at pH 7.2. An undifferentiated tubule. The tubule i s i n an early stage of development. The lumen (L) cont- aining some c e l l u l a r debris (arrow) i s s t i l l small. A tubule consisting of a single layer of c e l l s i s probably formed from the growth and movement of such a group of undifferentiated c e l l s . Then these undifferentiated c e l l s acquire the u l t r a s t r u c t u r a l features c h a r a c t e r i s t i c of each portion of the nephron. The deposition of reaction p r e c i - pitate i s abundant a l l along the l a t e r a l membranes separating each i n d i v i d u a l c e l l . There i s also some pre c i p i t a t e on the luminal surface membranes. Only two of the n u c l e i (N) appear to have f i n e precipitate associated with them. x 5,700 81. Figure 55 2 hour-old kidney. 2 hours pre-fixation i n gluta- raldehyde. No pre-incubation exposure to L- cysteine. Incubation with L-cysteine at pH 7.2. An undifferentiated tubule. Occasionally a c i l i u m (arrow) i s seen a r i s i n g from the a p i c a l plasma membrane. Otherwise the luminal plasma membrane i s simple i n contour. A piece of cytoplasmic material ( X ) appears to be extruded into the lumen (L). Only the l a t e r a l membranes between various c e l l s show an abundant deposition of reaction p r e c i p i t a t e . x 16,500 82. Figure 56 2 hour-old kidney. 2 hours pre-fixation i n gluta- raldehyde. No pre-incubation exposure to L- cysteine. Incubation with L-cysteine at pH 7.2. An immature glomerulus. The podocytic c e l l s (p) are s t i l l c l o s e l y packed together. The cyto- plasm of some of the c e l l s are beginning to dev- elop into foot processes. There i s intense enzy- matic reaction on p r a c t i c a l l y a l l the membranes whether they are i n apposition or are free. Some of the free membranes show a les s e r amount of or no prec i p i t a t e (arrows). In one podocytic c e l l (pi) there i s also some prec i p i t a t e i n the cytoplasm. x 7,500 83. Figure 57 Brush border of a proximal tubule.The brush border (b) i s very well-developed. The micro- v i l l i are long and slender and are c l o s e l y packed together. This collapsed condition of the brush border i s a r t i f a c t u a l and i s a res u l t of the mode of f i x a t i o n used. The tubular i n - vaginations from the bases of the m i c r o v i l l i appear to contain a dense material (arrows). There are no a p i c a l v e s i c l e s or vacuoles i n the a p i c a l cytoplasm of t h i s p a r t i c u l a r c e l l . There i s no accumulation of electron-dense precipitate except f o r a few specks here and there. x 9,100 8 4 . Figure 5 8 D i s t a l tubule. The basal membranes i n f o l d ex- tensively d i v i d i n g the cytoplasm into numerous compartments within which are contained elongated mitochondria (M). The e x t r a c e l l u l a r compartments are enlarged s l i g h t l y probably i n response to f i x a t i o n conditions (arrows). On the luminal surface of the c e l l there are a few short m i c r o v i l l i (m). In the a p i c a l cytoplasm there are a few small a p i c a l v e s i c l e s (a). There i s no prec i p i t a t e on the basal i n t e r d i g i t a t i o n s , on the m i c r o v i l l i or within the cytoplasm. x 7,500 85. A glomerulus. The lumens of three c a p i l l a r i e s (C) are seen i n t h i s electron micrograph. The endothelium (e) l i n i n g the c a p i l l a r i e s i s fen- estrated. A thick basement membrane (bm) i s interposed between the endothelium and the podo- c y t i c foot processes ( f ) , which are cytoplasmic prolongations of the podocytic c e l l s (p). The endothelium, the basement membrane and the podo- c y t i c foot processes are the three components of the f i l t r a t i o n apparatus. No prec i p i t a t e i s observed within the glomerulus. x 9,100 86. Figure 40 A proximal tubule. The brush border (b) i s well- developed. In the a p i c a l cytoplasm there are a large number of small a p i c a l v e s i c l e s (a) and large a p i c a l vacuoles (v). Numerous mitochondria (M) are present i n the cytoplasmic compartments formed by the elaborate i n t e r d i g i t a t i o n s of the basal membranes. The e x t r a c e l l u l a r compartments are enlarged i n response to the conditions of f i x a t i o n . Some electron-dense pre c i p i t a t e i s observed i n the region of the brush border (compare with Figures 1 and IS). x 7,500 87 Figure 41 An undifferentiated tubule. The tubular c e l l s are u l t r a s t r u c t u r a l l y simple. The n u c l e i (N") are large. In the cytoplasm there a few small round mitochondria (M). The luminal surface membrane i s simple i n contour. Part of the a p i c a l cytoplasm seems to oe blebbing o f f (arrow) and being discarded into the lumen (L) as debris. Eo precipitate i s present on any of the plasma membranes. x 9,100 88. Figure 42 Brush "border of a developing proximal tubule. The brush border (b) i s r e l a t i v e l y well- developed. There are a large number of long slender m i c r o v i l l i . Tubular invaginations ( t ) , small a p i c a l v e s i c l e s (a) and large a p i c a l vacuoles (v) are present i n the a p i c a l cyto- plasm. There are also a few mitochondria ( M ) . There i s no precipitate present. x 11,800 89. Figure 45 An immature glomerulus. The p a r i e t a l epithelium (E) i s the outermost component of the glomerulus. Within Bowman's space (L) are the podocytic c e l l s (p). A large nucleus i s present i n the a p i c a l poles of the c e l l s . Podocytic foot processes (f) ari s e from the basal portions of the c e l l s and abutt onto the basement membrane separating the podocytes from the endothelium (e). The cap- i l l a r y lumen (C) i s s t i l l small. There i s no deposition of precipitate i n the glomerulus. (The dark chunks are d i r t p a r t i c l e s ) . x 4,700 90. Figure 44 A developing proximal tubule. The m i c r o v i l l i (m), though s t i l l few i n number, are long and slender. Tubular invaginations ( t ) , small a p i c a l v e s i c l e s (a) and large a p i c a l vacuoles (v) are present i n the a p i c a l cytoplasm. There are quite a few mitochondria (M), but these appear to be p a r t i c u l a r l y susceptible to the effects of f i x - ation. Electron-dense precipitate i s seen adhering to the m i c r o v i l l i membranes and some of the tub- u l a r invaginations and a p i c a l v e s i c l e s . The amount of pr e c i p i t a t e i s less than i n 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 a c t i v i t y i n herring g u l l s a l t glands. J. Histochem. OytOchem. 17:570-584 (1969). Anderson, W.A.: Cytochemistry of sea urchin gametes I Intramitochondrial l o c a l i s a t i o n of glycogen, glucose-6-pMsphatase and ATPase a c t i v i t y i n spermatozoa of Paracentrotus l i v i d u s . J . U l t r a s t r u c t . Res. 24:398-411 (1968). Ashworth, C.T., Luibel, P.J. and Stewart, S.C.: The fi n e s t r u c t u r a l l o c a l i s a t i o n of ATPase on the small in t e s t i n e , kidney and l i v e r of the r a t . J . C e l l B i o l . 17:1-13 (1963). Barden, H. and Lazarus, S.S.: Histochemical character- i s t i c s of ATP-dephosphorylating enzymes i n rabbit pancreas. J . Histochem. Cytochem. 11:578-589 (1965). Barden, H. and Lazarus, S.S.: Some histochemical character i s t i c s of ATPase of mouse s t r i a t e d muscle. J . Histochem. Cytochem. 12:11-12 (1964). Barrnett, R.J.: The demonstration with the electron microscope of the end products of histochemical reactions i n r e l a t i o n to the fi n e structure of c e l l s . E x p tl! C e l l Res. Suppl. 7:65-89 (1959). Berg, G.G.: The staining of triphosphatases by the chelate removal method. J. Histochem. Cytochem. 12:341-358 (1964). Bonting, S.L., Caravaggio, L.L. and Hawkins, N.M.: Studies on sodium-potassium activated ATPase IV Correlation with cation transport sensitive to cardiac glycosides. Arch. Biochem. Biophys. 98:413-419 (1962). Bradshaw, M., Wachstein, M., Spence, J . and E l i a s , J.M.: ATPase a c t i v i t y i n melanocytes and epidermal c e l l s of human skin. • J . Histochem. Cytochem. 11:465-473 (1963). Brooke, M.H. and Kaiser, K.K.: Some comments on the h i s t o - chemical characterisation of muscle ATPase. J. Histochem. Cytochem. 17:431-432 (1969). 92. Bulger, R.E.: The shape of rat kidney tubular c e l l s . Am. J . Anat. 116:237-256 (1965). C a u l f i e l d , J.B. and Trump, B.F.: Correlation of u l t r a s t r - ucture with function i n rat kidney. Am. J . Pathol. 40:199-218 (1962). Clark, S.L.: C e l l u l a r d i f f e r e n t i a t i o n i n the kidney of newborn mice studied with the electron micros- cope. J. Biophys. Biochem. Cytol. 3:349-361 (1957). Deane, H.W.: Nuclear l o c a l i s a t i o n of phosphatase a c t i v i t y : fact or a r t i f a c t . J . Histochem. Cytochem. 11:443-444 (1963). Du Bois, A.M.: The embryonic kidney.Iln "The kidney Vol. I. Morphology, biochemistry, physiology." C. R o u i l l e r and A.F. Muller (eds.). Academic Press, New York. pp. 1-59 (1969). Ericsson, J.L.E.: Transport and digestion of hemoglobin i n the proximal tubule. I Light microscopy and cytochemistry of acid phosphatase. Lab. Investig. 14:1-15 (1965a). Ericsson, J.L.E.: Transport and digestion of hemoglobin i n the proximal tubule. II Electron microscopy. Lab. Investig. 14:16-39 (1965b). Ericsson, J.L.E. and Trump, B.F.: Electron microscopy of the u r i n i f e r o u s tubules. In "The kidney Vol. I. Morphology, biochemistry, physiology." C. R o u i l l e r and A.F. Muller (eds.). Academic Press, New York, pp. 351-447 (1969). Essner, E., Fogh, J . and Fabrizio, P.: L o c a l i s a t i o n of mitochondrial ATPase a c t i v i t y i n cultured human c e l l s . J . Histochem. Cytochem. 13:647-656 (1965). Essner, E., Novikoff, A.B. and Masek, B.: ATPase and 5- nucleotidase a c t i v i t i e s i n the plasma membrane of l i v e r c e l l s as revealed by electron microscopy. J. Biophys. Biochem. Cytol. 4:711-716 (1958). Fahimi, H.D. and Drochmans, P.: P u r i f i c a t i o n of g l u t a r a l - dehyde; i t s significance f o r preservation of acid phosphatase a c t i v i t y . J . Histochem. Cytochem. 16:199-204 (1968). 93. Farquhar, M.G. and Palade, G.E.: ATPase l o c a l i s a t i o n i n amphibian epidermis. J. C e l l B i o l . 30:359-379 (1966). Freiman, D.G. and Kaplan, N.: Studies on the histochemical d i f f e r e n t i a t i o n of enzymes hydrolysing ATP i n dog kidney. J. Histochem. Cytochem. 7:296 (1959). Freiman, D.G. and Kaplan, N.: Studies on the histochemical d i f f e r e n t i a t i o n of enzymes hydrolysing ATP. J. Histochem. Cytochem. 8:159-170 (i960). Gauthier, G.F.: On the l o c a l i s a t i o n of sarcotubular ATPase a c t i v i t y i n mammalian s k e l e t a l muscle. Histochemie 11:97-111 (1967). Gauthier, G.F. and Padykula, H.A.: Cytochemical studies of ATPase a c t i v i t y i n the sarcoplasmic reticulum. J. C e l l B i o l . 27:252-260 (1965). Glenner, G.G.: Enzyme histochemistry. In "Weurohistochem- i s t r y . " C.W.M. Adams (ed). E l s e v i e r Publishing Co., New York. pp. 109-160 (1965). Glenner, G.G.: Evaluation of the s p e c i f i c i t y of enzyme histochemical reactions. J . Histochem. Cytochem. 16:519-529 (1968). Goldfischer, S., Essner, E. and Novikoff, A.B.: The l o c a l - i s a t i o n of phosphatase a c t i v i t i e s at the l e v e l of u l t r a s t r u c t u r e . J . Histochem. Cytochem. 12:72-95 (1964). G r i f f i t h , L.D., Bulger, R.E. and Trump, B.F.: The u l t r a - structure of the functioning kidney. Lab. Investig. 16:220-246 (1967). Grossman, V/. and Heitkamp, D.H.: Electron microscopic l o c a l i s a t i o n of mitochondrial ATPase a c t i v i t y . J . Histochem. Cytochem. 16:645-653 (1968). H a l l , C.V.: Studies of normal glomerular structure by electron microscopy. 5th* Annual Conference on the Nephrotic Syndrome Vol. 5 pp. 1-39 (1953). H a l l , C.V.: The protoplasmic basis of glomerular u l t r a - f i l t r a t i o n . Am. Heart J . 54:1-9 (1957). Hoff, H.F. and Graf, J . : An electron microscopic study of phosphatase a c t i v i t y i n the endothelial c e l l of rabbit aorta. J . Histochem. Cytochem. 14:719-724 (1966). 94. Holt, S.J.: Factors governing the v a l i d i t y of staining methods f o r enzymes and t h e i r bearing upon the G-omori acid phosphatase technique. Exptl. C e l l Res. Suppl. 7:1-27 (1959). Holt, S.J. and Hicks, R.M.: Studies on formalin f i x a t i o n f o r electron microscopy and cytochemical s t a i n - ing purposes. J . Biophys. Biochem. Cytol. 11 : 3 1 - 4 5 . ( 1 9 6 1 ) . Hori, S.H. and Chang, J.P.: Histochemical study of ATPase i n cytoplasm. J . Histochem. Cytochem. 11:71-79 (1963). Jacobsen, N.O. and Jorgensen, P.L.: A quantitative biochem- i c a l and histochemical study of the lead method f o r l o c a l i s a t i o n of ATP-hydrolysing enzymes. J . Histochem. Cytochem. 17:443-453 (1969). Jacobsen, N.O., Jorgensen, F. and Thomsen, A.C.: On the l o c a l i s a t i o n of some phosphatases i n three d i f f e r e n t segments of the proximal tubules i n the rat kidney. J. Histochem. Cytochem. 15:456-469 (1967). Jones, D.B.: Mucosubstances of the glomerulus. Lab. Investig. 21:119-125 U 9 6 9 ) • Kaplan, S.E. and Novikoff, A.B.: The l o c a l i s a t i o n of ATPase a c t i v i t y i n rat kidney: electron microscopic examination of reaction product i n formal-calcium fixed frozen material. J . Histochem. Cytochem. 7:295 (1959). Kissane, J.M.: Quantitative histochemistry of the kidney I Segmental d i s t r i b u t i o n of enzymes i n the renal proximal tubule of normal r a t . J. Histochem. Cytochem. 9:578-584 (1961). K l e i n , R.L.: A histochemical and biochemical study of nuclear ATP hydrolysis i n embryo heart. J. Histochem. Cytochem. 14:669-680 (1966). Latta, H., Maunsbach, A.B. and Osvaldo, L.: The fin e structure of renal tubules i n cortex and medulla. In "Ultrastructure i n b i o l o g i c a l systems Vol. II - Ultrastructure of the kidney." A.J. Dalton and F. Haguenau (eds.). Academic Press, London, pp. 1-56 (1967). Lazarus, S.S. and Barden, H.: Histochemistry &n<i electron microscopy of mitochondrial ATPase. J . Histochem. Cytochem. 10:285-293 (1962). 95. Lazarus, S.S. and Barden, H.: Ultramicroscopic l o c a l i s - ation of mitochondrial ATPase. J. U l t r a s t r u c t l Res. 10:189-193 (1964). Lazarus, S.S. and Vethamany, V.G.: Relation of histochemical v i s u a l i s a t i o n of mitochondrial ATPase and ion uptake. J. C e l l B i o l . 27:57A (1965). Loewy, A.G. and Siekevitz, P.': The membrane system and the exchange:.of materials. In " C e l l structure and function." A.G. Loewy and P. Siekevitz (eds.). Holt, Rinehart and Winston Inc., Few York, pp. 194-208 (1963). Mao, K. and Nakao, K.: Variation of l o c a l i s a t i o n of AMPase and ATPase a c t i v i t i e s at the plasma membrane of human prosta t i c e p i t h e l i a l c e l l s : an electron microscopic study. J. Histochem. Cytochem. 14:203-204 (1966). • Marchesi, V.T.: Problems i n the l o c a l i s a t i o n of membrane bound enzymes by electron microscopic cyto- chemistry. In " B i o l o g i c a l properties of the mammalian'surface membrane." L.A. Manson (ed.). The Wistar I n s t i t u t e Press, Philadelphia, pp. 39-51 (1968). Matthiessen, M.E.: Enzyme histochemistry of the pre- n a t a l development of human deciduous teeth. I Alkaline phosphatase, acid phosphatase and unspecific AS-esterase. Acta anat. 63:523-544 (1966). Maunsbach, A.B.: The influence of d i f f e r e n t f i x a t i v e s and f i x a t i o n methods on the u l t r a s t r u c t u r e of rat kidney, proximal tubule c e l l s . I Comparison of d i f f e r e n t perfusion f i x a t i o n methods and of glutaraldehyde, formaldehyde and osmium tetroxide f i x a t i o n . J . U l t r a s t r u c t l Res. 15:242-282 (1966a). Maunsbach, A.B.: Observations on the segmentation of the proximal tubule i n the rat kidney. Comp- arison of r e s u l t s from phase contrast, fluorescence and electron microscopy. J. U l t r a s t r u c t . Res. 16:239-258 (1966b). Maunsbach, A.B., Madden, S.C. and Latta, H.: Variations i n f i n e structure of renal tubular epithelium under d i f f e r e n t conditions of f i x a t i o n . J . U l t r a s t r u c t . Res. 6:511-530 (1962). McClurkin, I.T.: A method f o r the cytochemical demon- 96. str a t i o n of sodium-activated ATPase. J . Histochem. Cytochem. 12:654-658 (1964). Moog, P.: Enzyme development i n r e l a t i o n to functional d i f f e r e n t i a t i o n . In "Biochemistry of animal development." R. Weber (ed.). Academic Press, New York. pp. 307-365 (1965). Moses, H.L. and Rosenthal, A.S.: On the significance of lead-catalysed hydrolysis of nucleoside phosphatases i n histochemical systems. J. Histochem. Cytochem. 15:354-355 (1967). Moses, H.L. and Rosenthal, A.S.: P i t f a l l s i n the use of lead ion f o r histochemical l o c a l i s a t i o n of nucleoside phosphatases. J. Histochem. Cytochem. 16:530-539 (1968). Moses, H.L., Rosenthal, A.S., Beaver, D.L. and Schuffman, S.S.: Lead ion and phosphatase histochemistry II Effect of ATP hydrolysis by lead ion on the histochemical l o c a l i s a t i o n of ATPase a c t i v i t y . J . Histochem. Cytochem. 14:702-710 (1966). Novikoff, A.B.: Enzyme l o c a l i s a t i o n with- Wachstein-Meisel procedures: r e a l or a r t i f a c t ? J . Histochem. Cytochem 15:353-354 (1967) Novikoff, A.B., Drucker, J . , Shin, W.Y. and Goldfischer, S.: Further studies of the apparent ATPase a c t i v i t y of c e l l membranes i n formol-calcium fixed tissues. J . Histochem. Cytochem. 9 : 4 3 4 - 4 5 1 (1961). Novikoff, A.B., Hausman, D.H. and Podber, E.: The l o c a l - i s a t i o n of ATPase i n l i v e r : i n s i t u s t aining and c e l l f r a c t i o n a t i o n studies. J . Histochem. Cytochem. 6:61-71 (1958). Ogawa, K. and Mayahara, H.: Intramitochondrial l o c a l i s a t i o n of ATPase a c t i v i t y . J . Histochem. Cytochem. 17:487-490 (1969). Otero-Vilardebo, L.R. , Lane, N. and Godman, G-.C: Demon- str a t i o n of mitochondrial ATPase a c t i v i t y i n formalin-fixed colonic e p i t h e l i a l c e l l s . J . C e l l B i o l . 19:647-652 (1963). Padykula, H.A. and Gauthier, G.F.: Cytochemical studies of ATPases i n s k e l e t a l muscle f i b r e s . J. C e l l B i o l . 18:87-107 (1963). Padykula, H.A. and Herman, E.: Factors a f f e c t i n g the a c t i v i t y of ATPase and other phosphatases as 97. measured by histochemical techniques. J. Histochem. Cytochem 3:161-167 (1955a). Padykula, H.A. and Herman, E.: The s p e c i f i c i t y of the histochemical method f o r ATPase. J. Histochem. Cytochem. 3:170-195 (1955b). Pease, D.C.: Pine structures of the kidney seen by electron microscopy. J. Histochem. Cytochem. 3:295-308 (1955). Per s i j n , J.P., Daems, W.T., DeMann, J.C.H. and Meijer, A.E.P.H. The demonstration of ATPase a c t i v i t y with the electron microscope. Histochemie 2:372-382 (1961). Pinkstaff, CA., Sandler, M. and Bourne, G.H.; Phosphatase studies on prenatal, neonatal and adult rat kidney. J. Geront. 17:267-271 (1962). Porter, K.R. and Bomneville, M.A.: "An introduction to the f i n e structure of c e l l s and t i s s u e s . " l e a and Febiger, Philadelphia. (1964). Post, R.L. and Sen, A.K.: An enzymatic mechanism of active sodium and potassium transport. J. Histochem. and Cytochem. 13:105-112 (1965). P r i e s t l y , G.C. and Molt, R.A.: Development of the meta- nephric kidney: protein and nucleic acid synthesis. J . C e l l B i o l . 37:703-715 (1968). Rechardt, L. and Kokko, A.: Electron microscopic ob- servations on the mitochondrial ATPase i n the rat s p i n a l cord. Histochemie 10:278-286 (1967). Rosenberg, T. and Wilbrandt, W.: Enzymatic processes i n c e l l membrane penetration. Int. Rev. Cytol. 1:65-92 (1952). Rosenthal, 0>S., Moses, H.L., Beaver, D.L. and Schuffman, S.S.: lead ion and phosphatase histochemistry I Non-enzymatic hydrolysis of nucleoside phos- phates by lead ion. J. Histochem. Cytochem. 14:698-701 (1966). Rosenthal, A.S., Moses, H.L., Tice, L. and Ganote, C.E.: Lead ion and phosphatase histochemistry. I l l The e f f e c t s of lead and ATP concentration on the incorporation of phosphate into fixed t i s s u e . J . Histochem. Cytochem. 17:608-612 (1969). 9 8 . R o u i l l e r , C : General anatomy and histology of the kidney. In "The kidney Vol. I. Morphology, biochemistry, physiology." C. R o u i l l e r and A.F. Muller (eds.). Academic Press, New York. pp. 61-156 ( 1 9 6 9 ) . Sabatini, D.D., Bensch, K. and Barrnett, R.J.: Cytochem- i s t r y and electron microscopy: the preservation of c e l l u l a r u l t r a s t r u c t u r e and enzymatic a c t i v i t y by aldehyde f i x a t i o n . J . C e l l B i o l . 1 7 : 1 9 - 5 8 ( 1 9 6 3 ) . Sabatini, D.D., M i l l e r , P. and Barrnett, R.J.: Aldehyde f i x a t i o n f o r morphological and enzyme h i s t o - chemical studies with the electron microscope. J. Histochem. Cytochem. 1 2 : 5 7 - 7 1 ( 1 9 6 4 ) . Sandler, M. and Bourne, G.H.: Intranuclear histochemical l o c a l i s a t i o n of ATPase. J. Histochem. Cytochem. 1 0 : 6 3 6 ( 1 9 6 2 ) . Simon, G.T. and Chatelanat, F.: Ultrastructure of the normal and pathological glomerulus. In "The kidney Vol. I. Morphology, biochemistry, physiol- ogy." C. R o u i l l e r and A.F. Muller (eds.)." Academic Press, New York. pp. 2 6 2 - 3 4 9 ( 1 9 6 9 ) . Sjostrand, F.S.: Electron microscopy of c e l l s and tissues Vol. I. Instrumentation and techniques. Academic Press, New York. ( 1 9 6 7 ) . Solomon, A.K.: Pumps i n the l i v i n g c e l l . Sc. Am. 207 :100-108 ( 1 9 6 2 ) . Spater, H.W., Novikoff, A.B. and Masek, B.: ATPase a c t i v i t y i n the c e l l membranes of kidney tubule c e l l s . J . Biophys. Biochem. Cytol. 4 : 7 6 5 - 7 7 0 ( 1 9 5 8 ) . Stumpf, P.K.: Adenosine triphosphate. Sc. Am. A p r i l 1 9 5 3 : 8 5 - 9 2 ( 1 9 5 3 ) . Suzuki, Y.: An electron microscopy of the renal d i f f e r - entiation I Proximal tubule c e l l s . J . E l e c t . Micros. 6 : 5 2 - 6 5 ( 1 9 5 8 ) . Tetas, M. and Lowenstein, J..M.: The effect of bivalent metal ions on the hydrolysis of ADP and ATP. Biochem. 2 : 3 5 0 - 3 5 7 ( 1 9 6 3 ) . Tewari, H.B. and Bourne, G.H.: Histochemical studies on the l o c a l i s a t i o n of ATPase i n the cerebellum of the r a t . J . Histochem. Cytochem. 11:246-257 ( 1 9 6 3 a ) . 99. Tewari, H.B. and Bourne, G.H.: Histochemical studies on the d i s t r i b u t i o n of ATPase i n the trigeminal ganglion c e l l s of the r a t . J. Histochem. Cytochem. 11:511-519 (1963b) Tice, L.W.: lead-adenosine triphosphate complexes i n adenosine triphosphatase histochemistry. J. Histochem. Cytochem. 17:85-94 (1969). Tisher, C C , Bulger, R.E. and Trump, B.P.: Human renal ul t r a s t r u c t u r e I Proximal tubule of healthy individuals, lab. Investig. 15:1357-1394 (1966). Torack, R.M. and Barrnett, R.J.: Nucleoside phosphatase a c t i v i t y i n membranous f i n e structures of neurons and g l i a . J . Histochem. Cytochem. 11:763-772 (1963). Torack, R.M. and Markey, M.H.: The characterisation of s p e c i f i c ATPase a c t i v i t y i n rat brain by means of combined biochemical and histochemical methods. J . Histochem. Cytochem. 12:12 (1964). Tormey, J.McD.: Significance of the histochemical demonstration of ATPase i n e p i t h e l i a noted f o r active transport. Nature 210:820-822 (1966). Trunjp, B.P. and Ericsson, J.I.E.: The e f f e c t of the f i x a t i v e solution on the u l t r a s t r u c t u r e of c e l l s and t i s s u e s . A comparative analysis with p a r t i c u l a r attention to the proximal cdhvaluted tubule of the rat kidney. lab. Investig. 14:1245-1323 (1965). Vethamany, V.G. and Lazarus, S.S.: U l t r a s t r u c t u r a l l o c a l - i s a t i o n of magnesium-dependent dinitrophenol- stimulated ATPase i n human blood p l a t e l e t s . J . Histochem. Cytochem. 15:267-272 (1967). Wachstein, M.: Histochemical staining reactions of the normally functioning and abnormal kidney. J. Histochem. CytQchem. 3:246-270 (1955). Wachstein, M. and Besen, M.: Electron microscopic l o c a l - i s a t i o n of phosphatase a c t i v i t y i n the brush border of the rat kidney. J. Histochem. Cytochem. 11:447-448 (1963). Wachstein, M. and Besen, M.: Electron micoscopic study i n several mammalian species of the reaction 100. product enzymatically liberated from ATP i n the kidney. Lab. Investig. 15:476-489'(1964). Wachstein, M. and Bradshaw, M.: Histochemical ATPase a c t i v i t y i n dark and l i g h t c e l l s of c o l l e c t i n g tubules i n mammalian kidney. Nature 194:299-300 (1962). Wachstein, M. and Bradshaw, M.: Histochemical l o c a l - i s a t i o n of enzyme a c t i v i t y i n the kidneys of three mammalian species during t h e i r post-natal development. J. Histochem. Cytochem. 15:44-56 (1965). Wachstein, M., Bradshaw, M. and O r t i z , J.M.: Histochemical demonstration of mitochondrial ATPase a c t i v i t y i n tissue sections. J. Histochem. Cytochem. 10:65-74 (1962). Wachstein, M. and Fernandez, C : Electron microscopic l o c a l i s a t i o n of nucleoside triphosphatase i n endoplasmic reticulum of l i v e r and pancreas. J. Histochem. Cytochem. 12:40-42 (1964). Wachstein, M. and Meisel, E.: Histochemistry of hepatic phosphatases at a physiologic pH with s p e c i a l reference to the demonstration of b i l e c a n a l i c u l i . Am. J. C l i n . Pathol. 27:15-25 (1957). Wachstein, M., Meisel, E. and Niedzwiedz, A.: Histochem- i c a l demonstration of mitochondrial ATPase with the lead-ATP technique. J. Histochem. Cytochem. 8:587-388 (i960). Wacker, SIR., Zarkowsky, H.S. and Burch, H.B.: Changes i n kidney enzymes of rats a f t e r b i r t h . Am. J. Physiol. 200:567-569 (1961). Wheeler, K.P. and Whittam, R.: Structural and enzymic aspects of hydrolysis of ATP by membranes of kidney cortex and erythrocytes. Biochem. J . 95:549-363 (1964). Wills, E.J.: L o c a l i s a t i o n f o r electron microscopy of nucleoside phosphatases i n human l i v e r . J . Histochem. Cytochem. 15:754-755 (1967).- Wllmer, H.A.: The c o r r e l a t i o n between the f u n c t i o n a l a c t i v i t y of the renal tubule and i t s phosphatase content. Arch. Pathol. 37:227-257 (1944). 101. Wilson, P.D.: Electon microscopic demonstration of two types of mitochondria with d i f f e r e n t a f f i n i t i e s f o r lead. Histochem. J . 1 : 4 0 5 - 4 1 6 ( 1 9 6 9 ) . Yamada, E.: The f i n e structure of the renal glomerulus of the mouse. J . Histochem. Cytochem. 3 : 3 0 9 ( 1 9 5 5 ) . Yasuzumi, G. and Tsubo, I.: The f i n e structure of n u c l e i as revealed by electron microscopy III ATPase a c t i v i t y i n the pores of nuclear envelope of mouse choroid plexus e p i t h e l i a l C G I,,, 1 ^ Exptl.' C e l l Res. 4 3 : 2 8 1 - 2 9 2 ( 1 9 6 6 ) .

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
China 3 25
United States 1 0
City Views Downloads
Beijing 2 0
Shenzhen 1 25
Ashburn 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}

Share

Share to:

Comment

Related Items