and Villa-Santal found no evidence that hyperplasia ... - Europe PMC

THE AMERICAN JOURNAL OF PATHOLOGY VOLUME XLIX

JULY, I966

NUMBER I

COMPENSATORY RENAL ENLARGEMENT HYPERTROPHY VERSUS HYPERPLASIA HORTON A. JOHNSON, M.D.,

AND

J. M. VERA ROMAN *

From the Medical Research Center, Brookhaven National Laboratory, Upton, N. Y.

The compensatory growth of the remaining kidney following unilateral nephrectomy has, over the years, been referred to alternatively as hypertrophy or hyperplasia, but the relative importance of these processes is a long-standing and still unresolved question. Since the investigations of Arataki I and Moore 2 it has been an accepted fact that mature nephrons can increase in size but not in number, and in this sense compensatory renal enlargement is purely hypertrophic. At the cellular level, however, there has been a variance of opinion. Initially it was thought that renal epithelium, like neurons, could not be stimulated to divide and that nephrons could increase in size only as a result of cellular hypertrophy. This concept dates back to I902, when Galeotti and Villa-Santal found no evidence that hyperplasia played any role in the enlargement of the adult kidney. Twenty-five years later Saphir,4 in a study of compensatory growth of the rabbit kidney, stated definitely that he saw no mitotic figures in glomeruli or renal tubular epithelium, and again attributed the renal enlargement wholly to cellular hypertrophy. This remained the prevailing opinion, as reflected by standard pathology textbooks of that time,5 until I949, when RollasonI demonstrated a brief burst of mitotic activity in young rats, chiefly in convoluted tubular epithelium, within the first few days after unilateral nephrectomy. In addition to this clear-cut cellular hyperplasia, Rollason saw an increase in size of epithelial cells and concluded that both cellular hypertrophy and hyperplasia contributed to compensatory renal growth. Research supported by the United States Atomic Energy Commission. Accepted for publication, March 3, 1966. * Present address: Facultad de Medicina, Universidad de Madrid, Madrid, Spain. I1

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JOHNSON AND VERA ROMAN

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This brief postoperative flurry of mitotic activity, which had long been overlooked by many earlier investigators, has been well established by many studies in recent years. Among the most detailed descriptions of this phenomenon are those given by Williams 7 and Goss and Rankin 8 both of whom determined mitotic indices in kidneys of mature rats at various times following unilateral nephrectomy and have found a delay of about 24 hours followed by a sharp peak in mitotic index at 40 to 48 hours and a rapid decline thereafter. Williams also showed a second, smaller peak in mitotic activity between the third and fourth days; this did not appear in Goss and Rankin's data. A great deal of effort has been directed toward defining the factors which regulate and modify the hyperplastic response,8'17 but whether chromosomal replication is stimulated by an increased functional load placed on the single kidney or whether it is controlled directly by an organ-specific humoral agent is still an open question. The experiments presented here were carried out for the purpose of determining which of the two processes, hypertrophy or hyperplasia, was the primary response to unilateral neprectomy and which response was greater. This was done by comparing the two processes in regard to i) time of onset following the stimulus, and 2) relative contribution to renal enlargement after the peak of hyperplastic activity. Hyperplasia was measured by the fraction of cells in the phase of DNA synthesis at a given time (index of DNA synthesis). This method was used, not because it was necessarily more accurate or more informative than the mitotic index, but simply because it was easier. The increase in renal mass was measured by the dry organ weights, by the rate of incorporation of cytidine into RNA, and by the rate of incorporation of leucine into protein. Hypertrophy was measured by the increase in renal mass in excess of that required by cell proliferation. MATERIAL AND METHODS Swiss mice of the inbred Brookhaven National Laboratory strain served as experimental animals. Although rats have been used almost exclusively in the study of renal hypertrophy and hyperplasia, mice were used in this study simply because of the io-fold economy of radioactively labeled precursors. Mature male animals 6 to 8 months of age were used in all experiments. Nephrectomies were carried out under ether anesthesia. In each case the left kidney was approached through a dorsal subcostal incision, the kidney was brought through the wound with a minimum of dissection, the hilum was tied off with a single suture, and the kidney was cut free with minimal bleeding. The controls were subjected to sham operation by delivering the left kidney through the wound and then returning it to the retroperitoneum. For the measurement of thymidine incorporation, the tritiated compound (rI c per mM) was injected intraperitoneally. Each animal received 2 pc per gm body weight, a dose more than adequate for autoradiography but with no significant radiation effect.18 Thirty minutes after injection the animals were killed by cervical

July., Z966

COMPENSATORY RENAL ENLARGEMENT

3

dislocation. The right kidneys were fixed in Io per cent formalin, and autoradiograms were prepared in the usual way using Eastman NTB2 liquid emulsion.19 Labeled cells were counted in Ioo high power fields in each kidney. Since the renal cortex is the principal site of both hyperplasia7 and hypertrophy,20 fields were limited to the cortex and were taken in a zigzag pattern across all cortical levels, although it was apparent that more cells were labeled in the outermost zone. The degree of labeling was expressed as the fraction of all cells scanned. The rates of incorporation of leucine into protein were measured by liquid scintillation counting of renal cortical tissue after the injection of the labeled amino acid. One hour before sacrifice each animal received o.s ,uc of 14C-leudne (0.24 c per mM) per gm body weight injected intraperitoneally. Anesthetized animals were killed by gradual exsanguination through tail vessels in order that the kidneys might be as ischemic as possible. Specimens of renal cortex weighing about 50 mg were blotted on filter paper and weighed. The tissue was then homogenized in a Potter-Elvehjem homogenizer in lo cc of Io per cent trichloroacetic acid (TCA). The homogenate and protein precipitate were spun down and resuspended in a second change of TCA, followed by a washing in o.i per cent non-radioactive leucine, and a final washing in acetone. The remaining precipitate was dried in a 600 C oven. The dry precipitate was then hydrolyzed in 3 ml of warm Hyamine® hydroxide, and the resulting solution was dissolved in I2 ml of scintillation liquid (0.3 per cent phenylbiphenyloxadiazole in xylene). Samples were counted in a Packard Tri-carb liquid scintillation spectrometer with automatic quench determination. Specimens in which total 14C activity was counted were placed in hyamine directly after weighing, dissolved, and counted as described. The uptake of cytidine was measured by liquid scintillation counting of the tritiated compound. One hour before sacrifice animals were given injections intraperitoneally of z uc of BH-cytidine per gm of body weight (specific activity, I.IS c per mM). Specimens of renal cortex, about So mg each, were blotted, weighed, and hydrolyzed in warm hyamine hydroxide. The resulting solution was mixed with scintillation liquid and counted as described above. Kidney weights were determined by blotting the fresh kidneys and drying them in a vacuum chamber until constant weights were obtained. Dried kidneys were weighed on a ioo-mg torsion balance. RESULTS

The extent of DNA synthesis in the remaining kidney at various times after unilateral nephrectomy is shown in Text-figure I. The graph depicts the fraction of convoluted tubular epithelial cell nuclei which were labeled by 3H-thymidine at a given time. It was felt that further subdivision of cell types was not necessary for the purposes of this study, although Williams,7 in his detailed analysis of mitotic indices, found a greater proliferative response in the proximal than in the distal convoluted tubules. The index of DNA synthesis of interstitial cells was also determined, but the results were so variable, even among animals in a single group, that they have not been included here. This variability of interstitial cell proliferation, perhaps a result of varying degrees of very low-grade chronic pyelonephritis, was so great in comparison with the low level of DNA synthesis among parenchymal cells that it could introduce a considerable error into overall determinations of DNA synthesis

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4

in the kidney. Initially an attempt was made to evaluate the proliferative component of compensatory renal growth by measuring the total DNA synthesis by means of injected '25I-iododeoxyuridine, using gamma ray -J

DNA

4

-JI z Ua.

0

2

RNA

PROTEIN

a2

3

24 36 12 48 HOURS AFTER NEPHRECTOMY TEXT-FIG. 4. Data of Text-figures I and 3 plotted on a relative scale for comparion.

constant size of cell population, No. That is to say, the control value of IL is that required to maintain a steady state. Any increase in number of cells engaged in DNA synthesis, NAI., will presumably result in an increase in the size of the cell population. Accordingly, the rate at which excess cells pass through the synthesis phase is equal to the rate at which

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Vol. 49, No. z

additional cells are added to the population. This statement can be written in the form of an equation: NAI_ dN dt' te where t. is the mean duration of the synthetic phase and dN d-t is the rate of increase in the size of the cell population. But AIl varies according to the time after nephrectomy so that AI = f(t). Substituting and separating variables, dN - f(t) dt. Integrating,

In Nt = ! ff(t) dt + In No, rt

and

Nt

exp No

(I)

=

ts~~~~

0

Since in this case f(t) is not known, the integral can be approximated by measuring the area under the experimental curve minus the area under the control curve in Text-figure i. The duration of the synthesis phase, t., like the duration of mitosis, seems to be more or less similar from one cell type to another, varying from siX 24 to eleven 25 hours. In the intestinal epithelium of the normal mouse t. is 7y2 hours,26 whereas durations of 7 to io hours have been measured in a mouse sarcoma27 and a mouse carcinoma,28 respectively. For the calculation of increase in population size, a value of 8 hours is arbitrarily chosen as a reasonably close estimate. If the true value is longer than 8 hours, then the true increase in cell population size is even less than that calculated. The increase in number of cells at various times, given by applying Eq. (i) to the curves of Text-figure i and using t. = 8 hours, is plotted in Text-figure 5. At the end of the third postoperative day the number of cells in the remaining kidney was increased by 4.4 per cent, and after the fifth day the increase was only 7.0 per cent. Comparing this with the increase in dry weight, clearly the great bulk of compensatory growth was accounted for by cell hypertrophy. Five days after nephrectomy, at a time when proliferative activity had passed its peak and was in decline, cell proliferation could account for only one-fourth of the increase in organ mass. The balance of 75 per cent of the increase was a result of

July, I966


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increase in the average mass of the individual cell. The mean increase in mass per cell, as can be seen in Text-figure 5, was about 25 per cent during the first 5 days after nephrectomy. Equation (i) is a general statement of the effect of changing rate of cell proliferation upon the population size. By using the duration of mitosis in place of t6 it can be applied to data on mitotic indices as well.

30

w cn w U z

20

10

0

60 120 HOURS AFTER NEPHRECTOMY TEXT-BIG. S. Increase in size of cell population calculated from Eq. (i) and plotted together with relative increases in dry weight to illustrate the relative extents of hyperplasia and hypertrophy.

Substituting into Eq. (i) the data for the mitotic response in the rat given by Goss and Rankin,8 and using a mean mitotic time of I hour, gives a 5.6 per cent increase in cell population after 3 days and a 6.8 per cent increase after 4 days. The relatively small net increase in cell population size, about 7 per cent in 5 days, helps to explain why, in spite of a 6-fold increase in mitotic figures, several investigators have been unable to show a significant increase in total DNA in the early phases of compensatory growth.2931 The onset of pure cellular hypertrophy without hyperplasia immediately following contralateral nephrectomy may be in part, at least, a simple "work hypertrophy" in response to an increased functional load.

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Vol. 49, No. t

For many years the excretion of urine has been considered to be the main functional load of the kidney, and in that sense the kidney would, under normal conditions, use only a fraction of i per cent of the energy supplied to it, leaving a large energy reserve. More recently, however, it has been pointed out that tubular reabsorption represents most of the functional load, and that the kidney works quite efficiently with a small margin of unused energy.82 This could tend to make the kidney sensitive to overloading by virtue of sudden changes in hemodynamics, glomerular filtration rate, and tubular reabsorption. It also suggests that experiments designed to test the "functional load" hypothesis of compensatory growth by diverting a ureter into the gut or peritoneum, causing the kidney to "re-excrete" the urine,812 do in fact create only a small change in tubular reabsorption and hence in total functional load. The clear-cut and no doubt significant interval between the onset of RNA and protein synthesis and the onset of DNA synthesis is thought provoking. Apparently the factors which control compensatory growth of the kidney, whatever they may be, do not act primarily by regulation of DNA synthesis but by regulation of RNA and protein synthesis. This implies a likelihood that in this case cell proliferation is secondary to cell hypertrophy, that a cell divides only if and when it reaches a certain critical size or perhaps a certain critical ratio of cytoplasm to nucleus. This would also tend to explain the curious fact that in the presence of a growth simulus involving the entire organ only a few scattered cells, less than i in ioo, respond by initiating DNA synthesis: the few cells which respond may represent the thin, upper tail of a cell size distribution curve which is being shifted by generalized cell hypertrophy. This recalls the long popular "critical mass" hypothesis for the triggering of DNA synthesis and cell division88 (Text-fig. 6). Although renal cell hyperplasia begins only after a certain degree of cell hypertrophy has been reached, this does not mean that cell mass is necessarily a trigger for the initiation of cell division. Proof of the "critical mass" hypothesis would require a demonstration that increased cell proliferation could be prevented in the post-nephrectomy kidney by suppressing cell hypertrophy. This type of relationship has been well demonstrated in the ameba, in which cell division can be delayed indefinitely by repeated amputation of cytoplasm.33 The regenerating liver shows a sequence of synthetic activity similar to that in the compensating kidney, with the onset of increased RNA and protein synthesis followed by a delayed onset of DNA synthesis and mitotic activity.17 An example of quite the opposite sequence is given, however, by the mouse spermatocyte. The newly formed cell begins to synthesize DNA almost immediately, during which time the cell volume

July, z966


II

and number of mitochondria remain nearly constant. Suddenly, following the termination of DNA synthesis, the cell begins to enlarge rapidly, increasing its volume about 8-fold in 8 days and increasing its comple-

CrIlical/ Mass for Cell Div/S/cln

4dass of Cytop/asm poer Ce/I TEXT-FIG. 6. Scheme, based on the "critical masss" hypothesis, which relates the early hypertrophy, the delayed onset of hyperplasia, and the small number of cells which divide in response to a generalized growth stimulus. If all cells respond primarily by increasing cytoplasmic mass, a small fraction of these cells will divide as the upper tail of the distribution curve enters the critical zone.

ment of mitochondria at a mean rate of about 3 per hour.34 Although the premeiotic spermatocyte is not strictly comparable to premitotic somatic cells, it tends to discount cell hypertrophy as a prerequisite for the initiation of DNA synthesis. The question of whether there is, in mammalian cells, a causal relationship between hypertrophy and hyperplasia or whether they are independently regulated processes has yet to be answered. SUMMARY After unilateral nephrectomy in the mouse, the compensatory growth of the remaining kidney is characterized by an increase in RNA and protein synthesis within the first hour. DNA synthesis remains unchanged for about i8 hours and then rises to a maximum at 48 hours. By the end of the fifth day, when DNA synthesis has passed its peak and is in decline, cellular hyperplasia has accounted for only one-fourth of the increase in kidney weight. Thus it is shown that cell hypertrophy is both the primary and the predominant response in the early phase of compensatory renal enlargement. REFERENCES I. ARATAKI, M. Experimental researches on the compensatory enlargement of the surviving kidney after unilateral nephrectomy (Albino rat). Amer. J. Anat., 1926, 36, 437-450.

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2. Mooim, R. A. The number of glomeruli in the kidney of the adult white rat unilaterally nephrectomized in early life. J. Exp. Med., I929, 50, 709-712. 3. GALEOTTI, G., and VILLA-SANTA, G. Ueber die compensatorische Hypertrophie der Nieren. Zeigler's Beitr. Path. Anat. Allg. Path., I902, 3I, I2I-142. 4. SAHIR, 0. The state of the glomerulus in experimental hypertrophy of the kidneys of rabbits. Amer. J. Path., 1927, 3, 329-342. BovD, W. A Text Book of Pathology. An Introduction to Medicine. Lea & S. Febiger, Philadelphia, I947, ed. 5, 1049 pp. 6. ROLLASON, H. D. Compensatory hypertrophy of the kidney of the young rat with special emphasis on the role of cellular hyperplasia. Anat. Rec., I949, I04, 263-285. 7. WILLAmS, G.E.G. Some aspects of compensatory hyperplasia of the kidney. Brit. J. Exp. Path., I961, 42, 386-396. 8. Goss, R. J., and RANKIN, M. Physiological factors affecting compensatory renal hyperplasia in the rat. J. Exp. Zool., I960, I45, 209-2I6. 9. BREuHAus, H. C., and MCJUNKIN, F. A. Effect of macerated kidney on the mitotic rate of kidney epithelium. Proc. Soc. Exp. Biol. Med., I932, 29, 894895. 10. SAETREN, H. A principle of auto-regulation of growth; production of organ specific mitose-inhibitors in kidney and liver. Exp. Cell Res., 1956, II, 229232.

I I.

I2.

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14.

Is. I6. I7.

I8. 19.

20.

OGAWA, K., and NowINSKI, W. W. Mitosis stimulating factor in serum of unilaterally nephrectomized rats. Proc. Soc. Exp. Biol. Med., I958, 99, 350354. SIMPSON, D. P. Hyperplasia after unilateral nephrectomy and role of excretory load in its production. Amer. J. Physiol., I96I, 201, 517-522. WILLmIAs, G.E.G. Studies on the control of compensatory hyperplasia of the kidney in the rat. Lab. Invest., I962, II, 1295-I302. LOWENSTEIN, L. M., and STERN, A. Serum factor in renal compensatory hyperplasia. Science, I963, I42, I479-1480. BENITEZ, L., and SHAKA, J. A. Cell proliferation in experimental hydronephrosis and compensatory renal hyperplasia. Amer. J. Path., I964, 44t 96I-972. SEMENOVA, N. F. [The role of the functional load during the process of cellular activation in regeneration of the kidney in mice.] Biull. Eksp. Biol. Med., I964, 57, IO8-III. Goss, R. J. Adaptive Growth. Academic Press, London, I964, 360 pp. JOHNSON, H. A., and CRONKITE, E. P. The effect of tritiated thymidine on mouse spermatogonia. Radiat. Res., I959, II, 825-831. JOHNSON, H. A., and BOND, V. P. A method of labeling tissues with tritiated thymidine in vitro and its use in comparing rates of cell proliferation in duct epithelium, fibroadenoma, and carcinoma of human breast. Cancer, I96I, I4, 639-643. MORRISON, A. B. Experimentally induced chronic renal insufficiency in the rat.

Lab. Invest., I962, II, 32I-332. 2I. FRANCK, G. ttude du metabolisme et de la synth6se des acides d6soxyribonucleiques au cours de l'hypertrophie compensatrice du rein chez le rat jeune, par cytophotometrie, caryom6trie et histoautoradiographie. Arch. Biol. (Liege), I960, 71, 489-525. 22. RoSEN, V. J., JR., and COLE, L. J. Radiosensitivity of mouse kidney undergoing compensatory hypertrophy. Nature (London), I960, I87, 6I2-6I4.

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23. ARGYRIS, T. S. Effect of unilateral nephrectomy or damage on mitotic activity in the contra-lateral kidney. (Abstract) Amer. Zool., I96I, I, 434. 24. PAINTER, R. B., and DREw, R. M. Studies on deoxyribonucleic acid metabolism in human cancer cell cultures (HeLa). Lab. Invest., I959, 8, 278-285. 25. BASERGA, R. Mitotic cycle of ascites tumor cells. Arch. Path. (Chicago), I963, 75, I56-I6I. 26. QUASTLER, H., and SHERMAN, F. G. Cell population kinetics in the intestinal epithelium of the mouse. Exp. Cell Res., I959, I7, 420-438. 27. JOHNSON, H. A. Some problems associated with the histological study of cell proliferation kinetics. Cytologia (Tokyo), I96I, 26, 32-41. 28. MENDELSOHN, M. L.; DOHAN, F. C., JR., and MooRE, H. A., JR. Autoradiographic analysis of cell proliferation in spontaneous breast cancer of C3H mouse. I. Typical cell cycle and timing of DNA synthesis. J. Nat. Cancer Inst., I960, 25, 477-484. 29. KENNEDY, G. C. Age and Renal Disease. In: Ciba Foundation Colloquia on Ageing. WOLSTENHOLME, G.E.W., and O'CONNOR, M. (eds.). J. & A. Churchill, Ltd., London, i958, Vol. 4, pp. 250-263. 30. MinADA, D. S., and KURNICK, N. B. Further studies on kidney growth: compensatory renal growth following unilateral nephrectomy in the rat. (Abstract) Fed. Proc., I960, I9, 325. 3 1. STRAUBE, R. L., and PATT, H. M. Effect of local x-irradiation on growth capacity of mouse kidney. Proc. Soc. Exp. Biol. Med., I96I, io8, 8o8-8io. 3 2. JOHNSON, H. A., and KNUDSEN, K. D. Renal efficiency and information theory. Nature (London), I965, 206, 930-93I. 33. MAZIA, D. The life history of the cell. Amer. Sci., I956, 44, I-32. 34. JOHNSON, H. A., and HAMMOND, H. D. The rate of mitochondrial increase in the murine spermatocyte. Exp. Cell Res. I963, 3I, 6o8-6io.