Histochemistry 46, 107--120 (1976) 9 by Springer-Verlag 1976
Morphometric and Enzyme Histochemical Behaviour of the Kidney of Young Rats before and after Unilateral Nephreetomy* J e r z y Kazimierczak, Pierre Chavaz, Radivoj Krstid, a n d Otto Bueher Institute of Histology and Embryology of the University of Lausanne, Switzerland Received September 18, 1975
Summary. Four week old male Wistar rats were unilaterally nephrectomized, and one, two, four, fourteen, and twenty-eight days after the operation the remaining kidneys were removed for morphological examination including measurement of diameters of the tub u l e s - a n d for histochemical investigation of enzymatic activities in the nephrons. The following enzymes were examined: suceinate DH, lactate DH, ~-glycerophosphate DH, isocitrate DH, glucose-6-phosphate DH, cytoehrome oxidase, adenosine triphosphatase, alkaline and acid phosphatases. In comparison with kidneys of control animals of the same age and sex, the hypertrophy of the remaining kidney was found to be, even in these young rats, entirely due to enlargement of preexisting nephrons. The measurements have indicated that the hypertrophy was most distinct between the second and fourth day after uninephreetomy. Four weeks after the operation the diameters of proximal and distal tubules were about 21% and 18% respectively bigger in the uninephrectomized animals than in the corresponding controls. This increase in size was accompanied by some changes in intensity of enzymatic staining reactions; after an initial diminishment, a graduate increase in activity of almost all the observed tubular enzymes was found; this seems to be the expression of the functional compensation by the remaining kidney. Introduction The effect of u n i l a t e r a l n e p h r e c t o m y has b e e n i n v e s t i g a t e d i n a large n u m b e r of species i n c l u d i n g m a n : a n e n l a r g e m e n t of t h e r e m a i n i n g k i d n e y was t h u s observed as t h e most characteristic a n d almost c o n s t a n t p h e n o m e n o n , generally referred to as a c o m p e n s a t o r y r e n a l h y p e r t r o p h y or a c o m p e n s a t o r y renal growth. There exists no u n a n i m i t y , however, if this increase i n size results from a cellular h y p e r t r o p h y , from a hyperp]asia or from both. Recently, even a n u m e r a l augm e n t a t i o n of n e p h r o n s in the r e m a i n i n g k i d n e y of u n i l a t e r a l l y nephrectomized rats has been suggested (Bonvalet et al., 1972; I m b e r t et al., 1974; Canter a n d Goss, 1975). I t is n o t completely settled if the physical r e m o v a l of the renal tissue or only the d i m i n i s h m e n t of t h e excretory f u n c t i o n stimulates t h e accelerated r e n a l growth; moreover, evidences were p r o v i d e d t h a t m a n y other factors m a y play a significant role i n the degree of the morphologically d e m o n s t r a t e d hypert r o p h y (see Nowinski a n d Goss, 1969). * Paper dedicated to Professor Dr. Drs. h. e. W. Bargmann (Kiel) in honour of the 70th anniversary of his birthday.
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The f u n c t i o n of the r e m a i n i n g k i d n e y during the t i m e of c o m p e n s a t o r y growth has likewise been largely discussed. Peters (1963) divided the period of functional c o m p e n s a t i o n i n (1) a c o m p e n s a t o r y a d a p t a t i o n a n d (2) a c o m p e n s a t o r y hyperf u n c t i o n characterised b y a raised glomerular f i l t r a t i o n rate i n the l a t t e r a n d a d i m i n i s h e d t u b u l a r sodium r e a b s o r p t i o n with increased rate of u r i n e flow i n the former. Variations i n the rate of glomerular filtration a n d of oxygen u p t a k e with only a n n n s i g n i f i c a n t g l o m e r u l o - t u b u l a r i m b a l a n c e has been observed i n the r e m a i n i n g k i d n e y also b y other a u t h o r s (Dicker a n d Shirley, 1971), thus i m p l y i n g metabolic changes occurring in the t u b u l a r cells. A c t u a l l y the biochemical observations of F a r q u h a r et al. (1968), K a t z a n d E p s t e i n (1967), a n d S c h m i d t a n d D u b a c h (1974) seem to corroborate such a n assumption, although some other reports dealing with t h e same subject have provided somewhat different i n f o r m a t i o n s (Dies a n d Lotspeich, 1967). H e n c e the p r e s e n t i n v e s t i g a t i o n was u n d e r t a k e n with i n t e n t to find out whether f u n c t i o n a l changes following u n i l a t e r a l n e p h r e c t o m y m a y be evidenced b y histochemical reactions for some oxidative a n d h y d r o l y t i c enzymes whose presence in the t u b u l a r cells is generally regarded as indispensable for t h e n o r m a l f u n c t i o n of the nephron. I n order to collect more i n f o r m a t i o n concerning the w a y a n d the degree of h y p e r t r o p h y i n the i n v e s t i g a t e d animals, t h e kidneys were also e x a m i n e d histologically, a n d diameters of t h e cortical t u b u l e s were measured.
Material and Methods Male Wistar rats, four weeks old, were used in all experiments. The left kidneys of 15 rats were excised through a dorsolateral incision of the body wall. The unilaterally nephrectomized and the control animals of the same age and weight were housed two or four in a cage at room temperature, and they received food and water ad libitum. One, two, four, fourteen, and twenty-eight days after the nephrectomy, the right kidneys of the operated and the control rats were removed. All the operations were performed under 5Tembutal anesthesia, and to avoid any circadian effects they were carried out at the same time of day, between 9:00 and 10:00 a.m. Small pieces of the experimental and the control kidneys were placed next to each other on a thin cork plate, rapidly frozen in isopentane chilled with carbon dioxide, and mounted in the cryostat. For the animals sacrified one and two days after the operation, the earlier removed left kidney (rapidly frozen and kept at --25 ~ C) served as an additional control. Besides, approximately 1 mm cubes of the same tissues were fixed for one hour in 2 % glutaraldehyde and 1% formaldehyde-mixture, postfixed for the same time in 1% osmium tetroxide and embedded in Durcupan. Enzyme histochemistry was performed on the fresh frozen 8-10 fzm thick cryostat sections. The close apposition of the experimental and control tissues made it possible to cut them simultaneously and at the same thickness. They were picked up side by side on the same slide and then identically treated during the histochemical procedure. This arrangement (Figs. 4 and 6) greatly facilitated a relatively objective evaluation of alterations in the intensity of staining reactions in the observed tissues, if present. Following enzymes were investigated: succinate dehydrogenase (DH), lactate DH, ~-glycerophosphate DH (menadione reductase), isocitrate DH, and glucose-6-phosphate DH, using nitroblue tetrazolium as hydrogen acceptor (Pearse, 1972). The latter enzyme was also demonstrated with monotetrazolium (MTT) in the incubating medium. Burnston's method for cytochrome oxidase and Gomori's cMcium-cobalt method for alkaline phosphatase (Pearse, 1968) were used. Gomori's lead technique was also employed to demonstrate acid phosphatase (Barka and Anderson, 1962) and ATPase (Wachstein and Meisel, 1957). Both, unfixed and fixed sections, were investigated by the latter reaction. All the reactions were carried on
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at room temperature except those for acid phosphatase and glucose-6-phosphate DH wich were done at 37 ~ C. After the incubations, the sections were counterstained with 1% methylen green, mounted in glycerin-gelatine and studied in the microscope. The morphology of the hypertrophying kidney was observed in 1 ~m thick plastic sections stained with alkaline toluidine blue. The same sections served for measurement of the diameters of the cortical proximal and distal tubules. This was done in a light microscope with an oil-immersion objective (magnification • 85) and an eyepiece with inserted scale (magnification • 10). Approximately 50 measures of the proximal and distal tubules of each animal were taken; the results are represented in a graphical form as m e a n s • S.E. in Fig. 1. Although by this method the absolute values were not the same as in the living animals, relative comparisons between the experimental and control animals appeared to be valid. The evaluation of the intensity of the histochemical staining reactions as well as the measurements of the tubules were arranged in such a way that the investigator had no orientation of the origin of the observed material.
Results K i d n e y s of t h e u n o p e r a t e d r a t s a r e c o n s i d e r e d as control kidneys, a n d t h e r e m a i n i n g k i d n e y s in t h e u n i l a t e r a l l y n e p h r e c t o m i z e d a n i m a l s as experimental kidneys. T h e m a c r o s c o p i c c o m p a r i s o n of t h e e x p e r i m e n t a l a n d t h e c o n t r o l k i d n e y s h a s s h o w n , a l r e a d y w i t h i n a few d a y s f o l l o w i n g t h e r e m o v a l of o n e k i d n e y , that the remaining organ was distinctly enlarged.
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Fig. 2. Comparative distribution of the investigated enzymatic activities in the kidneys of yoiung control rats (graphically represented in schematic designs of a nephron) and, under neath, in the remaining kidneys of rats killed 1, 2, 4, 14, and 28 days after unilateral nephrectomy. The relative differences between the control and the experimental animals are indicated by --, (--), = , (+), and + signifying respectively quite decreased, little decreased, equal, little increased, and quite increased intensity of the histochemical reactions in the kidneys of the nephrectomized rats. JGA: juxtaglomerular (or epithelioid) and Goormaghtigh cells
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Fig. 3. Alkaline pho~phatase in the cortex of a control kidney. The reaction is positive in the brush border of the proximal tubules (Pr) and in the Goormaghtigh cells (arrow) of the juxtaglomerular apparatus. Gl glomeruhls; MD macula densa. Magnification • 650
Morphometric Investigations Light microscopical examination of the histological sections has revealed that the experimental kidneys in the early period of hypertrophy as well as in the more advanced one consisted entirely of well developed nephrons. The size of nephrons in these kidneys appeared somewhat bigger than in the control organs, but no morphological particularities suggesting another process than enlargement of the preexisting nephrons were found. The measurements have then indicated a progressive increase in diameter of the cortical tubules both in the not yet adult control and in the experimental animals, although in the latter this increase was much more distinct (Fig. 1). Four weeks after the nephrectomy, the diameters of proximal and distal convoluted tubules was about 22 and 18 per cent respectively bigger in the operated animals than in the corresponding controls. The increase was most prominent between the second and fourth day after the operation, and then the rate of further growth was not particularly different between these two groups of animals. Size differences between tubules of the experimental and control animals could also be observed in the outer zone of the medulla, particularly in its outer stripe (see Figs. 6-9).
Histochemieal Investigations Histoehemical examination of the kidneys taken from the four week old control rats showed all the nephrons in each kidney identically supplied with the studied enzymes, although the sites and intensities of the particular staining reactions in different segments of the nephrons depended upon the investigated
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Fig. 4. Isocitrate D H in the cortex of control (on the left side) and experimental kidneys (on the right side) four days after the operation; the arrow shows the limit between these two. Well stained tubules in both kidneys appear more reactive and somewhat bigger in the experimental animal. Magnification X 55
enzyme. Thus acid p h o s p h a t a s e was l o c a t e d in t h e big a n d small granules of t h e p r o x i m a l c o n v o l u t e d a n d s t r a i g h t portions, respectively, while alkaline p h o s p h a t a s e , a l t h o u g h also p r e s e n t in t h e same portions, was seen e n t i r e l y in t h e b r u s h border. The l a t t e r e n z y m e was also f o u n d in t h e G o o r m a g h t i g h cells of t h e j u x t a g l o m e r u l a r a p p a r a t u s (Fig. 3) a n d in t h e a d v e n t i t i a of t h e arteries.
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Fig. 5a and b. Adenosine triphosphatase in (a) unfixed and (b) formalin fixed (5 minutes at + 4 ~ C) frozen sections of the control rat. Gl glomerulus; D1 distal tubule; Pr proximal tubule; MD macula densa; A/afferent arteriole. Magnification X650
The d i s t r i b u t i o n of t h e o x i d a t i v e enzymes a n d A T P a s e in t h e n e p h r o n of t h e control animals is r e p r e s e n t e d in schematic f o r m in Fig. 2. T h e r e it is shown t h a t b o t h t h e p r o x i m a l a n d d i s t a l t u b u l e s are b e s t s u p p l i e d w i t h t h e s e enzymes. T h e reactions are g e n e r a l l y s t r o n g e r in t h e p r o x i m a l c o n v o l u t i o n t h a n in t h e succeeding s t r a i g h t p o r t i o n with t h e e x c e p t i o n of t h o s e for succinate D H (Fig. 7) 3 Histochemistry
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Fig. 6. ~-glycerophosphate D H in the kidney of a control (on the left side) and that of an experimental rat (on the right side) four days after unilateral nephrectomy. The histochemical reaction in the straight portion of the proximal tubules is stronger in the control than in the experimental kidney, while the tubular size in the latter appears bigger. Magnification • 55
and ~-glycerophosphate D H (Figs. 6 and 9) where the reverse is true. I n the distal tubule the initial part (pars medullaris) of the straight portion exhibits usually the strongest enzyme activity (Fig. 8); the glueose-6-phosphate DH, particularly with monotetrazolium MTT as h y d r o g e n acceptor, gives also a v e r y strong reaction in the cells of the maeula densa. These cells, however, in the reaction for succinate D H are v e r y weakly stained and this is also v a h d for the A T P a s e reaction, b u t only in unfixed sections (Fig. 5a); in fact the fixation
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Fig. 7 a and b. Succinate DH (a) in the kidney of a control and (b) in t h a t of an experimental r a t two weeks after unilateral nephrectomy. The differences in staining intensity are not well discernible at this magnification; the size a u g m e n t a t i o n of the tubules in the remaining kidney of the nephrectomized animal is apparent. Magnification • 40 Fig. 8a and b. Cytochrome oxidase (a) in the outer medulla of the kidney of a control and (b) in t h a t of an experimental r a t two weeks after unilateral nephrectomy. The size of the tubules and their enzymatic activity are greater in the remaining kidney. Magnification X 125
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Fig. 9a and b. ~-glycerophosphate D H (a) in the kidney of a control and (b) in that of an experimental rat four weeks after unilateral nephrectomy. The straight portion of the proximal tubules shows a stronger reaction in the remaining kidney of the nephrectomized animal. Note also the bigger diameter of these tubules and the larger renal cortex of the operated animal. Magnification • 40
changes entirely the pattern of ATPase both in the juxtaglomerular apparatus and in the whole nephron (Fig. 5b). The distribution of the investigated enzymes in the experimental kidneys corresponds to that observed in the controls. All the nephrons are well supplied with the enzymes and no local or general changes in the patterns can be found (Figs. 4, 7, and 9). On the other hand, certain differences in intensity of the reactions between tubules of the experimental and corresponding control kidneys are seen. The nephron population is uniformly affected and the differences are best distinguishable in the proximal and distal tubules, particularly in their straight portions (Figs. 6, 8, and 9). No change in the thin segments as well as in the glomeruli and the J G A is perceptible. The intensity variations of enzymatic staining reactions in the uninephrectomized rats in comparison to the control animals are summarized in Fig. 2. During the first days following uninephrectomy the enzymatic activity in the tubules generally decreases (Fig. 6).
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Then, about four days after the operation, succinate DII,isocitrate DH, lactate DH, and cytochrome oxidase increase in activity, showing an even stronger reaction in the initial part of the straight portion of the distal tubules than in the corresponding tubules of the controls (Fig. 8). The raised activity of these enzymes in the proximal tubules is apparent 10 days later when also the other dehydrogenases appear to be more active. In the experimental kidneys an augmentation of ATPase activity was observed first four weeks after the nephrectomy and that was visible predominately in the straight portion of the distal tubules. On the other hand, the reactions for alkaline and acid phosphatases exhibit increased activities in the same kidneys already the second day after the operation, and this persists throughout the experiment. Discussion
The distribution of the examined oxidative and hydrolytic enzymes in our control material corresponds in general to that described by other authors (Hess et al., 1958; Jasmin, 1967; Jacobsen et al., 1967; Gomba et al., 1967 ; KrompecherKiss et al., 1971; Jacobsen and Jorgcnsen, 1973). All the nephrons in our four week old control rats were well supplied with the enzymes and no zonal diminishment of activity suggesting immaturity of the kidney (Fisher and Gruhn, 1959; Strand and Wattenberg, 1962; Kazimierczak, 1963; Wachstcin and Bradshaw, 1965) was seen. The same uniform pattern of enzymatic activity was observed in the remaining kidney of unilaterally nephrectomized rats, and since the morphological appearance did not suggest any numeral alterations of the nephrons, all speculations upon neogenic processes occurring in the compensatory growing kidney of young animals (Bonvalet et al., 1972 ; Imbert et al., 1974; Canter and Goss, 1975) have to be revised. Several contributions based upon histological techniques have indicated the unchangeable number of glomeruli in the remaining kidney of unilaterally nephrectomized animals (Kittelson, 1917; Arataki, 1926; Kazimierczak et al., 1975). Indeed, for a formation of new nephrons, the nephronogenic blastema is indispensable, which disappears in the rats somewhere at the end of the first decade of extrauterine life (Kazimierczak, 1971; Larsson, 1975). The compensatory renal growth proceeds entirely by enlargement of preexisting ncphrons. In our experiment this was apparent within a few days after removal of the contralatcrM organ, although it was shown by numerous other authors that the processes of renal adaptive growth initiate already a few hours after the operation and last until the lost renal efficiency is adequately restored (Peters, 1963; Johnson and Vcra Roman, 1966; Karp etal., 1971). IIayslctt, Kashgarian and Epstein (1968) measured tubular diameters in snap-frozen hypertrophied rat kidneys and reported the volumes of proximal and distal tubules increasing 96% and 47% respectively, about 2-4 weeks after the unilateral nephrectomy was performed. In our experimental animals the increase of tubular diameter (therefore also of the volume) was equally distinct in the proximal and distal tubules, with somewhat more rapid growth in the former (Fig. 1). Although our experimental techniques were different, the rats being much
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younger and thus the compensatory renal growth possibly different than in the older animals (Phillips and Leong, 1967; Verzs and IItigin, 1957; K a r p et al., 1971) also used by Hayslett and collaborators (1968), both studies indicate a clear enlargement of the renal tubules in the course of compensatory hypertrophy. This is true not only in the proximal but also in the distal tubules where at the same time a particularly augmented reabsorption of sodium takes place (Hayslett et al., 1968). The increase in size and reabsorption activity of the tubules during the compensatory growth are associated with increased cellular metabolic processes catalyzed by several enzyme systems. Hence, the early increasing contents of DNA and RNA in the kidney adapting to the new circumstances (Karp et al., 1971; Dicker and Shirley, 1973) were accompanied, according to Farquhar and collaborators (1968), by a progressive increase of hexose monophosphate shunt dehydrogenases, presumably providing pentose sugar indispensable for synthesis of these nueleotides. Furthermore, the augmented reabsorption of sodium (and other electrolytes) is connected with a rapid enhancement of activity of mierosomal Na K ATPase (Katz and Epstein, 1967; Sehmidt and Dubach, 1974) which appears to be especially integrated in the renal transmembrane transport. These observations based upon a quantitative measurement of enzymatic activities are not readily comparable with our histochemieal observations. During the first days following unilateral nephreetomy we observed in the remaining kidney an increased activity only for alkaline and acid phosphatases, while the other examined enzymes in the same organ appeared unchanged or even less active. I t should be considered, however, that our results qualify the intensity of staining reaction, not regarding the volumetric enlargement of the tubules. Making allowances for the latter factor, the final estimation of enzymatic activities per tubular segment seen in our histoehemieal reactions will change. This will be significant not only in the late phase of the hypertrophy, when we found a general increase of enzymatic activities in the experimental kidney, but also much earlier, when the differences in intensity of enzymatic staining reactions between the experimental and control materials were not distinct. I t m a y be concluded then, that in the remaining kidney of unilaterally nephreetomized young rats already within a few days following the operation there is a higher activity of some hydrolytic and oxidative enzymes discernible also b y histoehemical methods. This enhancement is presumably indispensable for augmented secretion and reabsorption of the remaining kidney stimulated to more or less double its habitual performances.
References Arataki, M. : Experimental researches on the compensatory enlargement of the surviving kidney after unilateral nephrectomy (Albino rat). Amer. J. Anat. 36, 437-450 (1926) Barka, T., Anderson, 1). J.: Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochem. Cytochem. 10, 741-753 (1962) Bonvalet, J.-P., Champion, M., Wanstok, F., Berjal, G.: Compensatory renal hypertrophy in young rats: Increase in the number of nephrons. Kidney Intern. 1, 391-396 (1972) Canter, Ch.E., Goss, R.J.: Induction of extra nephrons in unilaterally nephrectomized immature rats. Proc. Soc. exp. Biol. (N.Y.) 148, 294-296 (1975)
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Dicker, S.E., Shirley, D.G.: Rates of oxygen consumption and of anaerobic glycolysis in renal cortex and medulla of adult and new-born rats and guinea-pigs. J. Physiol. (Loud.) 2]2, 235-243 (1971) Dicker, S.E., Shirley, D.G.: Compensatory renal growth after unilateral nephrectomy in the new-born rat. J. Physiol. (Loud.) 228, 193-202 (1973) Dies, F., Lotspeich, W.D.: Hexose monophosphate shunt in the kidney during acid-base and electrolyte imbalance. Amer. J. Physiol. 212, 61-7l (1967) Farquhar, J . K . , Scott, W.N., Coe, F . L . : Hexose monophosphate shunt activity in compensatory renal hypertrophy. Proc, Soc. exp. Biol. (N.Y.) 129, 809-812 (1968) Fisher, E.R., Gruhn, J. : Maturation of succinate dehydrogenase and cytochrome oxidase in neonatal rat kidney. Proc. Soc. exp. Biol. (N.Y.) 101, 781-784 (1959) Gomba, Sz., Soltdsz, B.M., Szokoly, V.: Studies on the histochemistry of phosphatase enzymes in the juxtaglomerular complex. Histochemie 8, 264-274 (1967) Hayslett, J . P . , Kashgarian, M., Epstein, F . H . : Functional correlates of compensatory renal hypertrophy. J. clin. Invest. 47, 774-782 (1968). Hess, R., Scarpelli, D. G., Pearse, A. G.E.: The cytochemical localization of oxidative enzymes. II. Pyridine nucleotide-linked dehydrogenases. J. biophys, biochem. Cytol. 4, 753-760 (1958) Imbert, M. J., Berjal, G., Moss, N., de Rouffignac, C., Bonvalet, J. P. : Number of nephrons in hypertrophic kidneys after unilateral nephrectomy in young and adult rats. A functional study. Pflfigers Arch. 346, 279 290 (1974). Jacobsen, N. O., Jorgensen, F.: Further enzyme histochemical observations on the segmentation of the proximal tubules in the kidney of the male rat. Histochemie 34, 11-32 (1973) Jacobsen, N. O., Jorgensen, F., Thomsen, A. C.: On the localization of some phosphatases in three different segments of the proximal tubules in the rat kidney. J. Histochem. Cytochem. 15, 456-469 (1967) Jasmin, G.: Histochimie enzymatique du nephron chez le rat. Acta histochem, (Jena) 26, 160-168 (1967) Johnson, H. A., Vera Roman, J . M . : Compensatory renal enlargement. Hypertrophy versus hyperplasia. Amer. J. Path. 49, 1-13 (1966) Karp, R., Brasel, J.A., Winick, M.: Compensatory kidney growth after uninephrectomy in adult and infant rats. Amer. J. Dis. Child. 121, 186-188 (1971) Katz, A. I., Epstein, F. H. : The role of sodium-potassium-activated adenosine triphosphatase in the reabsorption of sodium by the kidney. J. clin. Invest. 46, 1999-2011 (1967) Kazimierczak, J. : Histochemical study of oxidative enzymes in rabbit kidney before and after birth. Acta anat. (Basel) 55, 352-369 (1963) K~zimierczak, J.: Development of the renal corpuscle and the juxtaglomerular apparatus. Acta path. microbiol, scand, suppl. 218 (1971) Kazimierczak, J., Chavaz, P., Krsti6, R., Bucher, O. : Some morphological and histochemical observations of the remaining kidney in unilaterally nephrectomizcd rats. Acta anat.. (Basel) in press (1975) Kittelson, J. A. : The postnatal growth of the kidney of the albino rat, with observations on an adult human kidney. Anat. Rec. 13, 385-406 (1917) Krompecher-Kiss, E., Bucher, 0., Krompecher, T. : t3ber das Verhalten einiger Oxydasen und Dehydrogenasen im juxtaglomerul~ren Apparat der Rattenniere. Morph. Jb. I16, 241--249 (1971) Larsson, L. : The ultrastructurc of the developing proximal tubule in the rat kidney. J. Ultrastruct. Res. 51, 119-139 (1975) Nowinski, W.W., Goss, R. J. (eds.): Compensatory renal hypertrophy. New York-London: Academic Press 1969 Pearse, A. G. E.: Histochemistry, theoretical and applied. 3rd ed. London: Churchill Ltd. 1968 (Vol. I) and 1972 (Vol. II) Peters, G. : Compensatory adaptation of renal functions in the unanesthetized rat. Amcr. J. Physiol. 205, 1042-1048 (1963) Phillips, T. L., Leong, G. F. : Kidney cell proliferation after unilateral nephrectomy as related to age. Cancer Res. 27, 286-292 (1967)
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SBhmidt, U., Dubach, U. C.: Induction of Na K ATPase in the proximal and distal convolution of the rat nephron after uninephrectomy. Pflfigers Arch. 346, 39-48 (1974) Strand, P. J., Wattenberg, L. W. : Coenzyme Q and succinate-tetrazolium reduetase activity of neonatal rat kidney. PROB. SoB. exp. Biol. (N.Y.) I I I , 230-233 (1962) VerzAr, F., Hfigin, F.: Einflu6 des Alters auf die Entwicklung der Arbeitshypertrophie yon Organen. Aeta anat. (Basel) 30, 918-927 (1957) Waehstein, M., Bradshaw, M. : Histoehemieal localization of enzyme activity in the kidneys of three mammalian species during their postnatal development. J. Histochcm. Cytochem. 13, 44-56 (1965) Waehstein, M., Meisel, E. : Histoehemistry of hepatic phosphatases at a physiological pH with special reference to the demonstration of bile Banaliculi. Amer. J. clin. Path. 27, 13 23 (1957) Dr. J. Kazimierczak Dr. P. Chavaz Dr. R. Krsti6 Prof. Dr. O. Bucher Institute of Histology and Embryology Rue du Bugnon 9 CH-1011 Lausanne Switzerland
Morphometric and Enzyme Histochemical Behaviour of the Kidney of Young Rats before and after Unilateral Nephreetomy* J e r z y Kazimierczak, Pierre Chavaz, Radivoj Krstid, a n d Otto Bueher Institute of Histology and Embryology of the University of Lausanne, Switzerland Received September 18, 1975
Summary. Four week old male Wistar rats were unilaterally nephrectomized, and one, two, four, fourteen, and twenty-eight days after the operation the remaining kidneys were removed for morphological examination including measurement of diameters of the tub u l e s - a n d for histochemical investigation of enzymatic activities in the nephrons. The following enzymes were examined: suceinate DH, lactate DH, ~-glycerophosphate DH, isocitrate DH, glucose-6-phosphate DH, cytoehrome oxidase, adenosine triphosphatase, alkaline and acid phosphatases. In comparison with kidneys of control animals of the same age and sex, the hypertrophy of the remaining kidney was found to be, even in these young rats, entirely due to enlargement of preexisting nephrons. The measurements have indicated that the hypertrophy was most distinct between the second and fourth day after uninephreetomy. Four weeks after the operation the diameters of proximal and distal tubules were about 21% and 18% respectively bigger in the uninephrectomized animals than in the corresponding controls. This increase in size was accompanied by some changes in intensity of enzymatic staining reactions; after an initial diminishment, a graduate increase in activity of almost all the observed tubular enzymes was found; this seems to be the expression of the functional compensation by the remaining kidney. Introduction The effect of u n i l a t e r a l n e p h r e c t o m y has b e e n i n v e s t i g a t e d i n a large n u m b e r of species i n c l u d i n g m a n : a n e n l a r g e m e n t of t h e r e m a i n i n g k i d n e y was t h u s observed as t h e most characteristic a n d almost c o n s t a n t p h e n o m e n o n , generally referred to as a c o m p e n s a t o r y r e n a l h y p e r t r o p h y or a c o m p e n s a t o r y renal growth. There exists no u n a n i m i t y , however, if this increase i n size results from a cellular h y p e r t r o p h y , from a hyperp]asia or from both. Recently, even a n u m e r a l augm e n t a t i o n of n e p h r o n s in the r e m a i n i n g k i d n e y of u n i l a t e r a l l y nephrectomized rats has been suggested (Bonvalet et al., 1972; I m b e r t et al., 1974; Canter a n d Goss, 1975). I t is n o t completely settled if the physical r e m o v a l of the renal tissue or only the d i m i n i s h m e n t of t h e excretory f u n c t i o n stimulates t h e accelerated r e n a l growth; moreover, evidences were p r o v i d e d t h a t m a n y other factors m a y play a significant role i n the degree of the morphologically d e m o n s t r a t e d hypert r o p h y (see Nowinski a n d Goss, 1969). * Paper dedicated to Professor Dr. Drs. h. e. W. Bargmann (Kiel) in honour of the 70th anniversary of his birthday.
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The f u n c t i o n of the r e m a i n i n g k i d n e y during the t i m e of c o m p e n s a t o r y growth has likewise been largely discussed. Peters (1963) divided the period of functional c o m p e n s a t i o n i n (1) a c o m p e n s a t o r y a d a p t a t i o n a n d (2) a c o m p e n s a t o r y hyperf u n c t i o n characterised b y a raised glomerular f i l t r a t i o n rate i n the l a t t e r a n d a d i m i n i s h e d t u b u l a r sodium r e a b s o r p t i o n with increased rate of u r i n e flow i n the former. Variations i n the rate of glomerular filtration a n d of oxygen u p t a k e with only a n n n s i g n i f i c a n t g l o m e r u l o - t u b u l a r i m b a l a n c e has been observed i n the r e m a i n i n g k i d n e y also b y other a u t h o r s (Dicker a n d Shirley, 1971), thus i m p l y i n g metabolic changes occurring in the t u b u l a r cells. A c t u a l l y the biochemical observations of F a r q u h a r et al. (1968), K a t z a n d E p s t e i n (1967), a n d S c h m i d t a n d D u b a c h (1974) seem to corroborate such a n assumption, although some other reports dealing with t h e same subject have provided somewhat different i n f o r m a t i o n s (Dies a n d Lotspeich, 1967). H e n c e the p r e s e n t i n v e s t i g a t i o n was u n d e r t a k e n with i n t e n t to find out whether f u n c t i o n a l changes following u n i l a t e r a l n e p h r e c t o m y m a y be evidenced b y histochemical reactions for some oxidative a n d h y d r o l y t i c enzymes whose presence in the t u b u l a r cells is generally regarded as indispensable for t h e n o r m a l f u n c t i o n of the nephron. I n order to collect more i n f o r m a t i o n concerning the w a y a n d the degree of h y p e r t r o p h y i n the i n v e s t i g a t e d animals, t h e kidneys were also e x a m i n e d histologically, a n d diameters of t h e cortical t u b u l e s were measured.
Material and Methods Male Wistar rats, four weeks old, were used in all experiments. The left kidneys of 15 rats were excised through a dorsolateral incision of the body wall. The unilaterally nephrectomized and the control animals of the same age and weight were housed two or four in a cage at room temperature, and they received food and water ad libitum. One, two, four, fourteen, and twenty-eight days after the nephrectomy, the right kidneys of the operated and the control rats were removed. All the operations were performed under 5Tembutal anesthesia, and to avoid any circadian effects they were carried out at the same time of day, between 9:00 and 10:00 a.m. Small pieces of the experimental and the control kidneys were placed next to each other on a thin cork plate, rapidly frozen in isopentane chilled with carbon dioxide, and mounted in the cryostat. For the animals sacrified one and two days after the operation, the earlier removed left kidney (rapidly frozen and kept at --25 ~ C) served as an additional control. Besides, approximately 1 mm cubes of the same tissues were fixed for one hour in 2 % glutaraldehyde and 1% formaldehyde-mixture, postfixed for the same time in 1% osmium tetroxide and embedded in Durcupan. Enzyme histochemistry was performed on the fresh frozen 8-10 fzm thick cryostat sections. The close apposition of the experimental and control tissues made it possible to cut them simultaneously and at the same thickness. They were picked up side by side on the same slide and then identically treated during the histochemical procedure. This arrangement (Figs. 4 and 6) greatly facilitated a relatively objective evaluation of alterations in the intensity of staining reactions in the observed tissues, if present. Following enzymes were investigated: succinate dehydrogenase (DH), lactate DH, ~-glycerophosphate DH (menadione reductase), isocitrate DH, and glucose-6-phosphate DH, using nitroblue tetrazolium as hydrogen acceptor (Pearse, 1972). The latter enzyme was also demonstrated with monotetrazolium (MTT) in the incubating medium. Burnston's method for cytochrome oxidase and Gomori's cMcium-cobalt method for alkaline phosphatase (Pearse, 1968) were used. Gomori's lead technique was also employed to demonstrate acid phosphatase (Barka and Anderson, 1962) and ATPase (Wachstein and Meisel, 1957). Both, unfixed and fixed sections, were investigated by the latter reaction. All the reactions were carried on
Morphometric and Enzyme Histoehemieal Behaviour after Nephrectomy
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PROXIMAL TUBULES 55. 5045 40-
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at room temperature except those for acid phosphatase and glucose-6-phosphate DH wich were done at 37 ~ C. After the incubations, the sections were counterstained with 1% methylen green, mounted in glycerin-gelatine and studied in the microscope. The morphology of the hypertrophying kidney was observed in 1 ~m thick plastic sections stained with alkaline toluidine blue. The same sections served for measurement of the diameters of the cortical proximal and distal tubules. This was done in a light microscope with an oil-immersion objective (magnification • 85) and an eyepiece with inserted scale (magnification • 10). Approximately 50 measures of the proximal and distal tubules of each animal were taken; the results are represented in a graphical form as m e a n s • S.E. in Fig. 1. Although by this method the absolute values were not the same as in the living animals, relative comparisons between the experimental and control animals appeared to be valid. The evaluation of the intensity of the histochemical staining reactions as well as the measurements of the tubules were arranged in such a way that the investigator had no orientation of the origin of the observed material.
Results K i d n e y s of t h e u n o p e r a t e d r a t s a r e c o n s i d e r e d as control kidneys, a n d t h e r e m a i n i n g k i d n e y s in t h e u n i l a t e r a l l y n e p h r e c t o m i z e d a n i m a l s as experimental kidneys. T h e m a c r o s c o p i c c o m p a r i s o n of t h e e x p e r i m e n t a l a n d t h e c o n t r o l k i d n e y s h a s s h o w n , a l r e a d y w i t h i n a few d a y s f o l l o w i n g t h e r e m o v a l of o n e k i d n e y , that the remaining organ was distinctly enlarged.
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Fig. 2. Comparative distribution of the investigated enzymatic activities in the kidneys of yoiung control rats (graphically represented in schematic designs of a nephron) and, under neath, in the remaining kidneys of rats killed 1, 2, 4, 14, and 28 days after unilateral nephrectomy. The relative differences between the control and the experimental animals are indicated by --, (--), = , (+), and + signifying respectively quite decreased, little decreased, equal, little increased, and quite increased intensity of the histochemical reactions in the kidneys of the nephrectomized rats. JGA: juxtaglomerular (or epithelioid) and Goormaghtigh cells
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Fig. 3. Alkaline pho~phatase in the cortex of a control kidney. The reaction is positive in the brush border of the proximal tubules (Pr) and in the Goormaghtigh cells (arrow) of the juxtaglomerular apparatus. Gl glomeruhls; MD macula densa. Magnification • 650
Morphometric Investigations Light microscopical examination of the histological sections has revealed that the experimental kidneys in the early period of hypertrophy as well as in the more advanced one consisted entirely of well developed nephrons. The size of nephrons in these kidneys appeared somewhat bigger than in the control organs, but no morphological particularities suggesting another process than enlargement of the preexisting nephrons were found. The measurements have then indicated a progressive increase in diameter of the cortical tubules both in the not yet adult control and in the experimental animals, although in the latter this increase was much more distinct (Fig. 1). Four weeks after the nephrectomy, the diameters of proximal and distal convoluted tubules was about 22 and 18 per cent respectively bigger in the operated animals than in the corresponding controls. The increase was most prominent between the second and fourth day after the operation, and then the rate of further growth was not particularly different between these two groups of animals. Size differences between tubules of the experimental and control animals could also be observed in the outer zone of the medulla, particularly in its outer stripe (see Figs. 6-9).
Histochemieal Investigations Histoehemical examination of the kidneys taken from the four week old control rats showed all the nephrons in each kidney identically supplied with the studied enzymes, although the sites and intensities of the particular staining reactions in different segments of the nephrons depended upon the investigated
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Fig. 4. Isocitrate D H in the cortex of control (on the left side) and experimental kidneys (on the right side) four days after the operation; the arrow shows the limit between these two. Well stained tubules in both kidneys appear more reactive and somewhat bigger in the experimental animal. Magnification X 55
enzyme. Thus acid p h o s p h a t a s e was l o c a t e d in t h e big a n d small granules of t h e p r o x i m a l c o n v o l u t e d a n d s t r a i g h t portions, respectively, while alkaline p h o s p h a t a s e , a l t h o u g h also p r e s e n t in t h e same portions, was seen e n t i r e l y in t h e b r u s h border. The l a t t e r e n z y m e was also f o u n d in t h e G o o r m a g h t i g h cells of t h e j u x t a g l o m e r u l a r a p p a r a t u s (Fig. 3) a n d in t h e a d v e n t i t i a of t h e arteries.
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Fig. 5a and b. Adenosine triphosphatase in (a) unfixed and (b) formalin fixed (5 minutes at + 4 ~ C) frozen sections of the control rat. Gl glomerulus; D1 distal tubule; Pr proximal tubule; MD macula densa; A/afferent arteriole. Magnification X650
The d i s t r i b u t i o n of t h e o x i d a t i v e enzymes a n d A T P a s e in t h e n e p h r o n of t h e control animals is r e p r e s e n t e d in schematic f o r m in Fig. 2. T h e r e it is shown t h a t b o t h t h e p r o x i m a l a n d d i s t a l t u b u l e s are b e s t s u p p l i e d w i t h t h e s e enzymes. T h e reactions are g e n e r a l l y s t r o n g e r in t h e p r o x i m a l c o n v o l u t i o n t h a n in t h e succeeding s t r a i g h t p o r t i o n with t h e e x c e p t i o n of t h o s e for succinate D H (Fig. 7) 3 Histochemistry
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Fig. 6. ~-glycerophosphate D H in the kidney of a control (on the left side) and that of an experimental rat (on the right side) four days after unilateral nephrectomy. The histochemical reaction in the straight portion of the proximal tubules is stronger in the control than in the experimental kidney, while the tubular size in the latter appears bigger. Magnification • 55
and ~-glycerophosphate D H (Figs. 6 and 9) where the reverse is true. I n the distal tubule the initial part (pars medullaris) of the straight portion exhibits usually the strongest enzyme activity (Fig. 8); the glueose-6-phosphate DH, particularly with monotetrazolium MTT as h y d r o g e n acceptor, gives also a v e r y strong reaction in the cells of the maeula densa. These cells, however, in the reaction for succinate D H are v e r y weakly stained and this is also v a h d for the A T P a s e reaction, b u t only in unfixed sections (Fig. 5a); in fact the fixation
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Fig. 7 a and b. Succinate DH (a) in the kidney of a control and (b) in t h a t of an experimental r a t two weeks after unilateral nephrectomy. The differences in staining intensity are not well discernible at this magnification; the size a u g m e n t a t i o n of the tubules in the remaining kidney of the nephrectomized animal is apparent. Magnification • 40 Fig. 8a and b. Cytochrome oxidase (a) in the outer medulla of the kidney of a control and (b) in t h a t of an experimental r a t two weeks after unilateral nephrectomy. The size of the tubules and their enzymatic activity are greater in the remaining kidney. Magnification X 125
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Fig. 9a and b. ~-glycerophosphate D H (a) in the kidney of a control and (b) in that of an experimental rat four weeks after unilateral nephrectomy. The straight portion of the proximal tubules shows a stronger reaction in the remaining kidney of the nephrectomized animal. Note also the bigger diameter of these tubules and the larger renal cortex of the operated animal. Magnification • 40
changes entirely the pattern of ATPase both in the juxtaglomerular apparatus and in the whole nephron (Fig. 5b). The distribution of the investigated enzymes in the experimental kidneys corresponds to that observed in the controls. All the nephrons are well supplied with the enzymes and no local or general changes in the patterns can be found (Figs. 4, 7, and 9). On the other hand, certain differences in intensity of the reactions between tubules of the experimental and corresponding control kidneys are seen. The nephron population is uniformly affected and the differences are best distinguishable in the proximal and distal tubules, particularly in their straight portions (Figs. 6, 8, and 9). No change in the thin segments as well as in the glomeruli and the J G A is perceptible. The intensity variations of enzymatic staining reactions in the uninephrectomized rats in comparison to the control animals are summarized in Fig. 2. During the first days following uninephrectomy the enzymatic activity in the tubules generally decreases (Fig. 6).
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Then, about four days after the operation, succinate DII,isocitrate DH, lactate DH, and cytochrome oxidase increase in activity, showing an even stronger reaction in the initial part of the straight portion of the distal tubules than in the corresponding tubules of the controls (Fig. 8). The raised activity of these enzymes in the proximal tubules is apparent 10 days later when also the other dehydrogenases appear to be more active. In the experimental kidneys an augmentation of ATPase activity was observed first four weeks after the nephrectomy and that was visible predominately in the straight portion of the distal tubules. On the other hand, the reactions for alkaline and acid phosphatases exhibit increased activities in the same kidneys already the second day after the operation, and this persists throughout the experiment. Discussion
The distribution of the examined oxidative and hydrolytic enzymes in our control material corresponds in general to that described by other authors (Hess et al., 1958; Jasmin, 1967; Jacobsen et al., 1967; Gomba et al., 1967 ; KrompecherKiss et al., 1971; Jacobsen and Jorgcnsen, 1973). All the nephrons in our four week old control rats were well supplied with the enzymes and no zonal diminishment of activity suggesting immaturity of the kidney (Fisher and Gruhn, 1959; Strand and Wattenberg, 1962; Kazimierczak, 1963; Wachstcin and Bradshaw, 1965) was seen. The same uniform pattern of enzymatic activity was observed in the remaining kidney of unilaterally nephrectomized rats, and since the morphological appearance did not suggest any numeral alterations of the nephrons, all speculations upon neogenic processes occurring in the compensatory growing kidney of young animals (Bonvalet et al., 1972 ; Imbert et al., 1974; Canter and Goss, 1975) have to be revised. Several contributions based upon histological techniques have indicated the unchangeable number of glomeruli in the remaining kidney of unilaterally nephrectomized animals (Kittelson, 1917; Arataki, 1926; Kazimierczak et al., 1975). Indeed, for a formation of new nephrons, the nephronogenic blastema is indispensable, which disappears in the rats somewhere at the end of the first decade of extrauterine life (Kazimierczak, 1971; Larsson, 1975). The compensatory renal growth proceeds entirely by enlargement of preexisting ncphrons. In our experiment this was apparent within a few days after removal of the contralatcrM organ, although it was shown by numerous other authors that the processes of renal adaptive growth initiate already a few hours after the operation and last until the lost renal efficiency is adequately restored (Peters, 1963; Johnson and Vcra Roman, 1966; Karp etal., 1971). IIayslctt, Kashgarian and Epstein (1968) measured tubular diameters in snap-frozen hypertrophied rat kidneys and reported the volumes of proximal and distal tubules increasing 96% and 47% respectively, about 2-4 weeks after the unilateral nephrectomy was performed. In our experimental animals the increase of tubular diameter (therefore also of the volume) was equally distinct in the proximal and distal tubules, with somewhat more rapid growth in the former (Fig. 1). Although our experimental techniques were different, the rats being much
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younger and thus the compensatory renal growth possibly different than in the older animals (Phillips and Leong, 1967; Verzs and IItigin, 1957; K a r p et al., 1971) also used by Hayslett and collaborators (1968), both studies indicate a clear enlargement of the renal tubules in the course of compensatory hypertrophy. This is true not only in the proximal but also in the distal tubules where at the same time a particularly augmented reabsorption of sodium takes place (Hayslett et al., 1968). The increase in size and reabsorption activity of the tubules during the compensatory growth are associated with increased cellular metabolic processes catalyzed by several enzyme systems. Hence, the early increasing contents of DNA and RNA in the kidney adapting to the new circumstances (Karp et al., 1971; Dicker and Shirley, 1973) were accompanied, according to Farquhar and collaborators (1968), by a progressive increase of hexose monophosphate shunt dehydrogenases, presumably providing pentose sugar indispensable for synthesis of these nueleotides. Furthermore, the augmented reabsorption of sodium (and other electrolytes) is connected with a rapid enhancement of activity of mierosomal Na K ATPase (Katz and Epstein, 1967; Sehmidt and Dubach, 1974) which appears to be especially integrated in the renal transmembrane transport. These observations based upon a quantitative measurement of enzymatic activities are not readily comparable with our histochemieal observations. During the first days following unilateral nephreetomy we observed in the remaining kidney an increased activity only for alkaline and acid phosphatases, while the other examined enzymes in the same organ appeared unchanged or even less active. I t should be considered, however, that our results qualify the intensity of staining reaction, not regarding the volumetric enlargement of the tubules. Making allowances for the latter factor, the final estimation of enzymatic activities per tubular segment seen in our histoehemieal reactions will change. This will be significant not only in the late phase of the hypertrophy, when we found a general increase of enzymatic activities in the experimental kidney, but also much earlier, when the differences in intensity of enzymatic staining reactions between the experimental and control materials were not distinct. I t m a y be concluded then, that in the remaining kidney of unilaterally nephreetomized young rats already within a few days following the operation there is a higher activity of some hydrolytic and oxidative enzymes discernible also b y histoehemical methods. This enhancement is presumably indispensable for augmented secretion and reabsorption of the remaining kidney stimulated to more or less double its habitual performances.
References Arataki, M. : Experimental researches on the compensatory enlargement of the surviving kidney after unilateral nephrectomy (Albino rat). Amer. J. Anat. 36, 437-450 (1926) Barka, T., Anderson, 1). J.: Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. J. Histochem. Cytochem. 10, 741-753 (1962) Bonvalet, J.-P., Champion, M., Wanstok, F., Berjal, G.: Compensatory renal hypertrophy in young rats: Increase in the number of nephrons. Kidney Intern. 1, 391-396 (1972) Canter, Ch.E., Goss, R.J.: Induction of extra nephrons in unilaterally nephrectomized immature rats. Proc. Soc. exp. Biol. (N.Y.) 148, 294-296 (1975)
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