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An autoradiographic study of the uptake of 3H-thymidine by kidney cells of mice at different ages
Experimental
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Cell Research
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AN AUTORADIOGRAPHIC STUDY OF THE UPTAKE 3H-THYMIDINE BY KIDNEY CELLS OF MICE AT DIFFERENT AGES1 RUTH Department
MARZANO
LITVAK
of Histology, Northwestern Northwestern University
University Medical
Received
April
(I 964)
OF
and R. BASERGAg Dental School,
School3 Chicago,
and Department Ill., U.S.A.
of Pathology,
5, 1963
THE use of aH-thymidine for labeling cells in DNA synthesis combined with autoradiography has provided a method for obtaining long term data concerning the proliferative activity of cells which have incorporated this DNA precursor. Cells which are undergoing DNA synthesis at the time of administration of the radioactive thymidine remain identifiable as labeled cells. In addition, inasmuch as the labeled thymidine of these cells will halve with each subsequent mitotic division, it becomes possible to determine the average duration of the intermitotic interval of these cells within a given period of time [9, 281. In the present study, age changes in these two features of 3H-thymidine labeling were examined in the mouse kidney. Mice of four ages, newborn to middle age, were given a single injection of 3H-thymidine and the changes with age in the proportion of cells synthesizing DNA synthesis at the time of injection examined in several histological regions of the kidney. Changes in the intensity of the label per cell between 1 and 18 days following the 3H-thymidine administration were also observed to determine variations with age in the average length of the intermitotic interval. With respect to the first of these observations, there is a continued increase in weight of the mouse kidney into old age; there is also evidence of a parallel increase in cell number for the organ as a whole [14]. From studies of the kidney of rat and man [l, 121, it is known that the increase in weight of this organ during aging is paralleled by an increase in volume of both the renal corpuscles and tubules. Whether cell proliferation contributes to both these increases remains 1 Research supported by USPHS Grant RG-6922 and in part by USPHS Grant 2 USPHS Research Career Development Awardee. s Present address: Department of Neurology, Northwestern University Medical Ill., U.S.A. Experimental
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C-5667. School,
Chic.,
3H-Thymidine
uptake
in kidney
cells
to be demonstrated. With respect to the length of the intermitotic interval of cells of the kidney, age related changes in this parameter have been investigated only in organs having a high rate of renewal [17]. MATERIALS
AND
METHODS
CAF, male mice, newborn, 2, 4 and 13 months old, bred under standardized laboratory conditions, were injected with 1 &gram body weight of W-thymidine (360 mc/mM in steriIe distilled water) obtained from Schwarz BioResearch Inc., Mt. Vernon, N.Y. This was administered subcutaneously to the newborn and intraperitoneally in the remaining age groups. Mice of each age group were killed one hour after injection of the labeled thymidine and at daily intervals therafter over an 18 day period. Tissue sections were prepared from kidney fixed in neutral formalin (10 per cent), embedded in paraffin and cut longitudinally through the hilum. The sections were cut at 5 p and prepared for autoradiography according to the stripping-film technique [21] with an exposure time of 30 days. After developing and fixing, the sections were stained with Mayer’s hematoxylin and eosin. Although the stripping-film technique gives good results for purposes of grain counting, its use results in a large loss of specimens [13] with a consequent reduction in sample size. Although it was intended to have two animals per interval for the 1 to 18 day period, the original sample size was greatly reduced. The final number of animals used is indicated in each of the graphs. The proportion of labeled to unlabeled cells was determined for each animal in all age groups on samples of 1000 to 3000 cells in each of two regions, the renal corpuscle, and the remaining part of the nephron tubule. In the newborn kidney, counts were also made in the nephrogenic zone. No distinction was made during counting between the glomerulus and its capsule since the proportion of labeled cells appeared to be equal in both parts of the renal corpuscle. In the region of the tubules, labeled cells in the cortex were found to exceed those in the medulla by four to five times in the three younger age groups, but in the kidney of the 13 month mouse, labeled cells were only rarely encountered in tubules within the medulla. There have been other reports of the relative paucity of labeling and of mitotic activity of cells within the medulla [19, 251, but in comparisons of the mitotic activity of cells of the outer and inner parts of the medulla in rats up to 2 months of age, the outer medulla almost equals the cortex in mitotic activity [24]. In the present study it was decided to randomize counts for the nephron tubules to include cells situated in the cortex and all of the medulla. In the nephrogenic zone, counts were randomized to include cells comprising its entire depth. The dilution of the label over an 18 day period was determined from mean grain counts obtained from samples of 50 labeled cells in each of the regions noted above. The individual grain counts per animal were also used in a few instances for the construction of histograms. The method of grain counting described by Kisieleski et al. [ll] was used. Only in the newborn was it necessary to compare grain counts across all cell types. These comparisons‘were facilitated by a similarity in the mean grain counts of cells of all three zones at 1 day following the uptake of the labeled thymidine. Experimental
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RESULTS
Labeling of cells of the renal corpuscle and the nephron tubule at 1 hr following the administration of 3H-thymidine in the newborn, 2, 4 and 13 month old mouse.-In Fig. 1 the number of labeled cells per thousand in the renal cor-
2
4
b
8
10
12
14
Age in months
Fig. l-Number of labeled cells per thousand in two regions of the mouse kidney at 1 hr following *H-thymidine injection, plotted against age. Each point represents mean counts from the following numbers of animals: two animals at the newborn interval, 6 animals at the 2- and at the 4month intervals, and 12 animals at the 13-month interval. The vertical bar (I) indicates *one standard deviation, l - l , cells of renal corpuscle; 0 - - 0, cells of nephron tubule.
puscle and tubule at 1 hr after the injection of labeled thymidine is plotted against age. At birth there is no difference between these regions in their incidence of labeling. Both regions show a sharp decline in labeling from birth to 2 months, and a continued but very slow decline from 2 to 13 months. The overall decline is of the same order for both regions, but at the 13-month interval, a 1 to 2 difference in labeling appears. When the standard deviations of the means at the 13-month interval (see Fig. 1) are used to assess this large difference, it is found to be statistically insignificant. Thus, in the kidney of the 13-month mouse, the number of cells in DNA synthesis is of the same order for the whole of the nephron tubule and for the whole of the renal corpuscle. And from birth to 13 months there appears to be no change in this relationship. Labeling of cells of the renal corpuscle and the nephron tubule from 1 to 18 days following aH-thymidine administration in the 2-, C- and 13-month-old Experimental
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mouse.-Approximately one specimen per age group per daily interval was retained for these observations. The percentage of labeled cells was found to fluctuate randomly within the 1 to 18 day period for all three age groups. The mean grain counts showed a similarly random fluctuation for this period. The absence of either an increase in labeled cells or of a decrease in h
240.5
Fig. 2.-Number of labeled cells per thousand in three regions of the kidney of the newborn mouse, from 1 to 16 days following a single injection of SH-thymidine. Each point represents a single animal. A, nephrogenic zone; o-0, renal corpuscle; 0 - - 0, nephron tubule.
the mean grain count of the labeled cells suggests that in these two regions of the 2- to 13-month mouse kidney the average time between two successive mitotic divisions is longer than 18 days. Labeling of cells in the nephrogenic zone, the renal corpuscle and the nephron tubule from 1 hr to 16 days following 3H-thymidine administration in the newborn mouse.-Changes in the proportion of labeled cells in three regions of the newborn kidney were observed between 1 hr and 16 days following a single injection of labeled thymidine (Fig. 2). Counts for the nephrogenic zone terminate at 7 days since this zone is no longer present after this time. From birth to 7 days new renal corpuscles and nephron tubules continue to be formed from cells of this zone by differentiation and migration [7, 83. In the present series, the nephrogenic zone at birth has a depth of approximately 70 cells and forms a considerable part of the kidney. These cells are arranged into tightly packed whorls which cap and occasionally surround the adjacent renal corpuscles and tubules. Between 1 hour and 1 day this zone undergoes a reduction in cell depth by approximately oneExperimental
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half. Reduction in depth and in compactness continue to the 7th day when the zone becomes visible only as discontinuous masses of cells of varying depth and arrangement. At the time of initial labeling, 1 hr post-injection, approximately 1 of 4 cells of the nephrogenic zone is labeled whereas a relatively small number, 1
Fig. S.-Mean grain counts in three regions of the kidney of the newborn mouse, from 1 to 16 days following a single injection of aH-thymidine. Each point represents a single animal. A-A, nephrogenic zone O-O, renal corpuscle, o - - 0, nephron tubule.
of 25, is labeled in the renal corpuscles and tubules. In view of these initial differences and the known fate of cells of the nephrogenic zone, it is to be expected that the counts of labeled cells of the renal corpuscle and tubule will rise continuously to the 7th day since for every four cells that they incorporate approximately one will be labeled. On the other hand, as the nephrogenic zone loses cells, no change in its proportion of labeled cells should occur, if cell loss occurs on a random basis. For this reason the points representing the daily counts for the nephrogenic zone were not connected in Fig. 2. Changes in the proportion of labeled cells between 1 hr and 16 days following thymidine administration include the following. In the nephrogenic zone there appears to be only random fluctuation during the first 3 days but a distinct lowering occurs between 4 and 7 days. The renal corpuscles and tubules show continuous rises in the proportion of labeled cells between 1 hr and 7 days. The increase between 1 hour and 1 day reflects largely the division of cells which are in DNA synthesis at the time of thymidine administration. The increase between 1 and 7 days will reflect acquisition of cells from the nephrogenic zone but it could also reflect, at the terminal part of the curve, a new division of labeled cells of the renal corpuscle and tubules Experimental
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present in these regions at birth or acquired sometime thereafter. Between 7 and 8 days both regions show an abrupt decline in labeled cells and this is followed by a second increase at lo-11 days which is maintained till the 16th day. The mean grain counts in the newborn (Fig. 3) were used to (1) determine
2
15 10 15 I
10
1
2 days
51J41LmLL 10
20
30
10
20
30
1 day 10 15 1
Fig. 4.-Number Each histogram days following
I
of grains per nucleus in cells of two regions of the kidney of the newborn mouse. is based on counts of 50 nuclei per region per animal killed at 1, 2, 7, 11 and 16 a single injection of labeled thymidine.
the average length of the intermitotic interval of cells of the nephrogenic zone, the renal corpuscle and the nephron tubule; (2) the source of newly labeled cells found in the renal corpuscle and tubules between 1 and 7 days and between 10 and 16 days following labeling, when the first and the second rise in labeled cells occurred. Mean grain counts for the nephrogenic zone show an approximate halving over the 1 day counts between 2 and 3 days, indicating the occurrence at this time of a mitotic division subsequent to the initial division which followed Experimental
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thymidine uptake. Thus, for cells of the nephrogenic zone labeled at birth, the intermitotic interval which follows the initial division has an average length of I to 2 days. Labeled cells which are still present in the nephrogenic zone between the 4th and 7th day do not show an additional decline in grain number. In the renal corpuscle and tubules, the mean grain counts are not appreciably altered from the first to the 4th day, when these regions showed a marked rise in labeled cells. Grain halving in these labeled cells occurs gradually and is not completed until the 1.Ith day. From this it may be assumed that a division subsequent to the initial division occurs approximately on the 1 lth day. Thus, for cells of the renal corpuscle and tubules labeled at birth, the intermitotic interval has an average length of approximately 10 days. It EolLows from these comparisons of mean grain counts that between 1 and 7 days the source of newly labeled cells found in the renal corpuscle and tubules is the labeled cell of the nephrogenic zone. This is supported by a gradual fall in the mean grain count in these two regions coincident with their gradual acquisition of cells of lower grain count from the nephrogenic zone. It follows also that the increase in labeled cells in these two regions between 31 and 16 days is to be attributed to a division of cells present in these regions at birth and labeled at the time of thymidine uptake. Histograms of the individual grain counts per specimen were also constructed (Fig. 4) to explore the possibility that cells of halved grain count acquired from the nephrogenic zone had, perhaps, undergone a second division, with a resulting second hiving of their grains and a consequent lowering of the mean grain counts of both the renal corpuscle and nephron tubule. The histograms do not support the possibility that any given segment of the population of labeled cells is exclusively involved in mitotic activity at 11-16 days post-injection. When the 11 and 16 day histograms are compared with the 1 and 2 day histograms, rather than finding a widening of the range, one finds an overall shift toward a reduced number of grains per cell, indicating that all cells are contributing to a lowering of the mean grain counts. A histogram of the individual counts at 7 days was included to demonstrate the widening of the range and the increase in incidence of cells of lower grain count which predictably follow the acquisition of cells of lowered grain count from the nephrogenic zone. The grain counts could be used only indirectly to account for the loss of labeled cells in the nephrogenic zone between 4 and 7 days and in the renal corpuscles and tubules between 7 and 8 days. In the first instance the loss at 4 days follows a mitotic division which results in a reduction of the mean Experimenlal
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grain count to six grains. With the ensuing loss of intensity of label per’nucleus, an artifactual loss of labeled cells may easily occur at the lower end of the range. The second instance of cell decline is of disturbingly large magnitude and possibly reflects a number of factors of which the most discernible include the following. With the artifactual loss of labeled nephrogenic cells, only 1 of 8 cells acquired at 7 days is a labeled cell. Sometime between 7 and 8 days the nephrogenic zone will be lost entirely as a source of cells whereas the unlabeled cells present in the renal corpuscle and tubule will continue to divide resulting in a decrease in the proportion of labeled cells. This decrease will continue until such time as the labeled cells are prepared to divide again. This doesn’t occur until the 1 lth day. Undue variation resulting from smallness of sample size at this critical time may also be a factor.
DISCUSSION
Studies of organ growth have been vastly broadened in scope by the introduction of a method for the labeling of cells and their subsequent identitication. Some of the many features of this method have been utilized in the present study of the mouse kidney in observing changes in the proliferative activity of several regions of this organ and in the proliferative activity of the labeled cells of these same regions over a relatively short period of time. Inasmuch as the percentage of cells labeled by 3H-thymidine has been shown to exceed the percentage of cells simultaneously observed in mitosis by 5 to 15 times [19, 261, the labeling method provides data on the proliferative activity of an organ of a larger order than can be obtained by the method of mitotic counts. This feature is especially useful in determining the proliferative activity of organs such as kidney in which mitotic activity decelerates so markedly with age that it is frequently thought to be absent. The additional feature of grain halving during successive divisions of labeled nuclei [2. 5, 9, 10, 28, 301 further provides data for estimating the average length of the intermitotic interval of labeled cells. In organs having a relatively slow growth this interval would otherwise be determinable only indirectly, from a consideration of the mitotic index and population size. In so far as the label remains, the method also provides a marker for migrating cells, as demonstrated in epiphyseal plate cartilage and periosteum [10, 291, in intestinal and esophageal epithelium [2, 16, 18, 20, 221 and in hemopoietic tissue (see [27]). In the present study this feature permitted observations of the rate at which the undifferentiated cells of the kidney of the newborn mouse differentiate and migrate to form renal corpuscles and nephron tubules. Experimental
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All age groups: proliferative activity in the renal corpuscle and nephron tubule.-The incidence of labeling at 1 hr following the administration of 3H-thymidine was found to decline with age for the renal corpuscle and nephron tubule. In the newborn a proliferation rate of 40 cells per thousand was obtained for each of these regions. From birth to 2 months these rates decline sharply to approximately three cells per thousand. Beyond 2 months a decline of much smaller magnitude occurs providing rates of approximately two cells per thousand at 4 months, and approximately one cell per thousand at 13 months. The magnitude of change among these older age groups approaches that found for periosteal cells of mice of similar ages [29]. When the frequencies of labeling in the renal corpuscle and tubules are plotted against age, one obtains for each region a curve that is inversely related to the cumulative growth curve of the kidney [14]. Thus one finds that, at 2 months, coincident with the beginning of the decelerated phase of growth of the whole body [23] as well as of the kidney, cell proliferation in two regions of the kidney has also entered a decelerated phase. Within the context of the present study it cannot be determined whether proliferative activity within the renal corpuscle and nephron tubule between 2 and 13 months will contribute to their own growth in volume, or whether this activity represents only population renewal by the numerical replacement of dying cells. It is the additive aspect of cell proliferation which tends to be emphasized when one considers that there is continued growth of the whole organ, which in itself reflects the continued growth of the glomeruli, the non-glomerular cortex and the medulla, as shown from studies of the kidney of the rat and man [1, 121. From findings that continued growth from early maturity into middle and old age of the non-glomerular cortex and medulla do not reflect a hypertrophy of the cells of the nephron tubule [ 11, it follows that growth of these two regions containing the tubules represents, in the aging animal, an increase in tubule length, and that this increase must be dependent upon the addition of new cells. For the glomeruli of the aging animal, increase in cell volume cannot be ruled out as a factor in glomerular growth [l] until data are obtained to indicate otherwise. But with the finding of continued proliferation of the renal corpuscle in the 2- to 13-month-old mouse, there is no need for minimizing the contribution of cell proliferation to the growth of the glomerulus of the aging animal. All age groups: length of the intermitotic interval of cells of three regions of the mouse kidney.-For the newborn, changes in the frequency of labeled cells and in the mean and individual grain counts were observed from 1 to 16 days following a single injection of 3H-thymidine. Cells of the renal corExperimental
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puscle and nephron tubule which are labeled at birth were found to have an intermitotic interval of approximately 10 days. For the labeled undifferentiated cell of the nephrogenic zone, the length of the intermitotic interval is approximately l-2 days. The subsequent mitotic interval of cells which remain in the nephrogenic zone is either of the same or of longer duration but could not be determined in the present series due, perhaps, to too great a dilution of the label. Intermitotic intervals for cells of other organs of the newborn have not been reported, although intervals of 14, 2 and 5 days have been reported for mesenchymal cells in three regions of the tibia of the g-dayold rat [30], and an interval of l& days has been reported for epithelial cells established in vitro from fetal calf liver [ 151. For the 2-, 4- and 13-month-old mouse, changes in the frequency of labeled cells and in the mean grain count were observed from 1 to 18 days following a single injection of 3H-thymidine. The counts remained randomly distributed during this interval, suggesting an intermitotic interval with a duration longer than 18 days. An intermitotic interval of 30-50 days, based on data relating to the mitotic index, has been reported for cells of the glomerulus and tubules of the kidney of the adult rat and mouse [26]. Further investigation will establish whether differences in this interval exist among the age groups used in the present study. The newborn: changes in the proportion of labeled cells in three regions of the kidney at 1 hr and from 1 to 16 days following a single injection of labeled thymidine.-This 16-day period is a period of rapid growth. Reportedly there is more than a doubling in size of the mouse kidney from birth to 2 weeks of age [4], and possibly the magnitude of this increase, if quantified, would approach that of the rat kidney where the volume doubles from birth to 3 days and doubles again from 3 to 10 days [ 11. At 1 hr following thymidine administration, 240 cells per thousand were found labeled in the nephrogenic zone. This exceeds by six times the number of cells labeled in the renal corpuscle and nephron tubule, where approximately 40 cells per thousand were found. Similar differences in the mitotic activity of these regions have been reported for the kidney of the rat taken just prior to term [25]. Cells of the very actively proliferating nephrogenic zone will contribute to the formation of new renal corpuscles and nephron tubules until the zone is exhausted of cells [S]. Dissolution of this zone occurs at 7-8 days in the present series. In the rat this occurs at lo-12 days [3] and at 10 days in the human [6]. It would thus appear that during the first postnatal week the nephrogenic zone provides the kidney with a source of cells far out of proportion to its own relative volume. Judging only from the extent to which labeled cells differenExperimental
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tiate between 1 hr and 3 days and again from 3 to 7 days it appears that a maximal contribution to kidney growth is made during the earlier period. From the rapid reduction in the depth of this zone at 1 day, the maximal contribution would appear to be made within the first day. However, this reduction in depth is accompanied by its circumferential growth as it keeps pace with the rapidly enlarging regions within. Changes in the number of labeled cells from 1 to 16 days following thymidine administration were otherwise observed primarily to corroborate the time of a second mitotic division of cells labeled at 1 hr following thymidine administration. In following these changes two periods of accumulation of labeled cells were found in the renal corpuscles and tubules. The first of these periods occurs from 1 to 7 days following thymidine administration. On the basis of grain counts this accumulation of labeled cells was attributed to the incorporation of labeled, newly formed renal corpuscles and tubules derived from the undifferentiated cells of the nephrogenic zone. In view of the fact that the renal corpuscles and tubules remain equally labeled from 1 to 7 days and that the renal corpuscles comprise only a small part of the kidney [ 121, it follows that the nephrogenic zone contributes numerically more cells to tubule formation than to the formation of renal corpuscles. It would thus appear that morphogenesis of the tubules at this time involves to a lesser extent the growth of the renal vesicle between its two poles [8] and to a greater extent a significantly large splitting away of presumptive renal vesicle cells. The second period of labeled cell accumulation occurs from 10 to 16 days following thymidine administration and follows, by several days, dissolution of the nephrogenic zone. On this basis and from a consideration of changes in the grain counts, this second accumulation of labeled cells was considered to be a consequence of a second division of renal corpuscle and tubule cells labeled at birth or of labeled cells acquired by these regions shortly after birth. CONCLUSIONS
Age related changes have been found in the proliferative activity of the renal corpuscle and nephron tubule regions and also in the length of the intermitotic interval of cells within these regions. From the newborn to the older age groups there is a decrease in proliferative activity of the order of 40 : 3 : 2 : 1, and there is an increase in the length of the intermitotic interval of undetermined magnitude. When the age factor is kept constant, no differences are found between these two regions. However, in the newborn, when these two regions are compared with a third region, the nephrogenic zone, large Experimental
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differences appear. The nephrogenic zone has a proliferative activity which exceeds that of the other two regions by six times, whereas the length of the intermitotic interval of the already differentiated cells exceeds that of the nephrogenic zone by approximately five to six times. In other studies dealing with variation with age in these two aspects of growth, a lengthening of the generation cycle by approximately one fifth has been found in intestinal epithelium [17], but age differences in the proliferative activity of this epithelium at zero time have not been found [16, IS]. The age groups used in these instances range from 3 to 31 months, overlapping the older age groups used in the present study and in a study of the proliferation of periosteal cells during aging [29]. In all these organs or tissues of older animals, the differences in proliferative activity tend to be of a small and possibly insignificant order. This suggests that the relationship between the number of cells found proliferating and the length of the intermitotic interval of these cells will tend not to be sharply defined in organs of older animals. In younger age groups in which regional variation in these two aspects of growth have been examined, differences have been reported for both the proliferative activity and length of the intermitotic interval in epiphyseal cartilage [lo] and mesenchymal cells [30] of the tibia. In the present study, in the two instances in which large differences in proliferative activity appear, where the kidney of the very young and old are compared, and where different regions of the newborn kidney are compared, it is significant that one finds an inverse relationship between the proliferative activity of the organ or region and the length of the intermitotic interval of its dividing cells. When one examines data given in the regional studies of cartilage and bone in the studies cited above, one finds that there is supporting evidence for this relationship.
SUMMARY
In the kidney of newborn, 2-, 4- and 13-month-old mice, the number of cells per thousand labeled after a single injection of 3H-thymidine declines with age. This decline is of the order of 40: 3:2: 1 for the cells of each of two regions, the renal corpuscle and the nephron tubule. Labeling of the nephrogenic zone of the newborn, which contributes cells for additional formation of the renal corpuscles and nephron tubules, was found to exceed by six times the labeling of each of the already differentiated regions. Intermitotic intervals were determined from grain counts recorded from Experimental
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0 to 18 days following a single injection of SH-thymidine; at 2, 4 and 13 months, this interval is longer than 18 days for cells of the renal corpuscle and nephron tubule; in the newborn it is ten days for these same cells and approximately 2 days for cells of the nephrogenic zone. Changes with age in proliferative activity are discussed with respect to known age changes in kidney volume. Regional differences in proliferative activity of the newborn kidney are discussed relative to the duration of the intermitotic interval of the cells of these regions and relative to the rate of differentiation of cells of the nephrogenic zone. REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
ARATAKI, M., Am. J. Anaf. 36, 399 (1926). BASERGA, R. and KISIELESKI, W. E., J. Natl. Cancer Inst. 28, 331 (1962). BAXTER, J. S. and YOFFEY, J. M., J. Anat. 82, 189 (1948). CLARK, JR., S. L., J. Biophys. Biochem. Cytol. 3, 349 (1957). EDWARDS, J. L., KOCH, A. L., YOUCIS, P., FREESE, H. L., LAITE, M. B., and DONALSON, J. T.: J. Biophys. Biochem. Cyfol. 7, 273 (1960). FELIX, W., The development of the urogenital organs. F. KEIBEL and F. P. MALL (eds.) Manual of Human Embryology, vol. 2 J. B. Lippencott Co. Phila. and London, 1912. HALL, B. V. and ROTH, L. E., Preliminary studies on the development and differentiation of cells and structures of the renal corpuscle. Electron Microscopy, Proceedings of the Stockholm Conference, 1956. HUBER, G. C., Renal tubules. E. V. COWDRY (ed.) Special Cytology, 2nd edition, p. 933. New York, Paul Hoeber, Inc., 1932. HUGHES, W. L., BOND, V. P., BRECKER, G., CRONKITE, E. P., PAINTER, R. B., QUASTLER, H. and SHERMAN, F. G., Proe. N&l. Acad. Sci. 44, 476 (1958). KEMBER, N. F., J. Bone and Joint. Surg. 42B, 824 (1960). KISIELESKI, W. E., BASERGA, R. and VAUPOTIC, J., Rad. 15, 341 (1961). KITTELSON, J. A., Anat. Rec. 13, 385 (1917). KOPRIWA. B. M. and LEBLOND, C. P., J. Hisfochem. Cyfochem. 10, 269 (1962). KURNICK, N. B. and KERNEN, R. L., J. Geronf. 17, 245 (1962). KUYPER, CH. M., SMETS, L. A. and PIECK, A. C. M., ExptZ Cell Res. 26, 217 (1962). LESHER, S., FRY, R. J. M. and KOHN, H. I., Lab. Invest. 10, 291 (1961). __ Gerontologia 5, 176 (1961). ( ~ Lab. Znuest. 11, 289 (1962). MESSIER, B. and LEBLOND, C. P., Am. J. Anat. 106, 247 (1960). QUASTLER, H. and SHERMAN, F. G., Exptt Cell Res. 17, 420 (1959). PELC, S. R., Znfern. J. Appf. Rad. Isotopes 1, 172 (1956). ~ Lab. Invest. 8, 225 (1959). ROBERTSON, T. B. J. Biot. Chem. 24, 363 (1916). ROLLASON, H. D., Anaf. Rec. 104, 263 (1949). __ Anat. Rec. 141, 183 (1961). SCHULTZE, B. and OEHLERT, W., Science 131, 737 (1960). STOHLMAN, F. (ed.), The Kinetics of Cellular Proliferation. New York, Grune and Stratton, 1959. TAYLOR, J. H., WOODS, P. S. and HUGHES, W. H., Proc. Nail. Acad. Sci. 43, 122 (1957). TONNA, E. A., J. Biophys. Biochem. Cytot. 9, 813 (1961). YOUNG, R. W., Exptl CeZZ Res. 26, 562 (1962).
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AN AUTORADIOGRAPHIC STUDY OF THE UPTAKE 3H-THYMIDINE BY KIDNEY CELLS OF MICE AT DIFFERENT AGES1 RUTH Department
MARZANO
LITVAK
of Histology, Northwestern Northwestern University
University Medical
Received
April
(I 964)
OF
and R. BASERGAg Dental School,
School3 Chicago,
and Department Ill., U.S.A.
of Pathology,
5, 1963
THE use of aH-thymidine for labeling cells in DNA synthesis combined with autoradiography has provided a method for obtaining long term data concerning the proliferative activity of cells which have incorporated this DNA precursor. Cells which are undergoing DNA synthesis at the time of administration of the radioactive thymidine remain identifiable as labeled cells. In addition, inasmuch as the labeled thymidine of these cells will halve with each subsequent mitotic division, it becomes possible to determine the average duration of the intermitotic interval of these cells within a given period of time [9, 281. In the present study, age changes in these two features of 3H-thymidine labeling were examined in the mouse kidney. Mice of four ages, newborn to middle age, were given a single injection of 3H-thymidine and the changes with age in the proportion of cells synthesizing DNA synthesis at the time of injection examined in several histological regions of the kidney. Changes in the intensity of the label per cell between 1 and 18 days following the 3H-thymidine administration were also observed to determine variations with age in the average length of the intermitotic interval. With respect to the first of these observations, there is a continued increase in weight of the mouse kidney into old age; there is also evidence of a parallel increase in cell number for the organ as a whole [14]. From studies of the kidney of rat and man [l, 121, it is known that the increase in weight of this organ during aging is paralleled by an increase in volume of both the renal corpuscles and tubules. Whether cell proliferation contributes to both these increases remains 1 Research supported by USPHS Grant RG-6922 and in part by USPHS Grant 2 USPHS Research Career Development Awardee. s Present address: Department of Neurology, Northwestern University Medical Ill., U.S.A. Experimental
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C-5667. School,
Chic.,
3H-Thymidine
uptake
in kidney
cells
to be demonstrated. With respect to the length of the intermitotic interval of cells of the kidney, age related changes in this parameter have been investigated only in organs having a high rate of renewal [17]. MATERIALS
AND
METHODS
CAF, male mice, newborn, 2, 4 and 13 months old, bred under standardized laboratory conditions, were injected with 1 &gram body weight of W-thymidine (360 mc/mM in steriIe distilled water) obtained from Schwarz BioResearch Inc., Mt. Vernon, N.Y. This was administered subcutaneously to the newborn and intraperitoneally in the remaining age groups. Mice of each age group were killed one hour after injection of the labeled thymidine and at daily intervals therafter over an 18 day period. Tissue sections were prepared from kidney fixed in neutral formalin (10 per cent), embedded in paraffin and cut longitudinally through the hilum. The sections were cut at 5 p and prepared for autoradiography according to the stripping-film technique [21] with an exposure time of 30 days. After developing and fixing, the sections were stained with Mayer’s hematoxylin and eosin. Although the stripping-film technique gives good results for purposes of grain counting, its use results in a large loss of specimens [13] with a consequent reduction in sample size. Although it was intended to have two animals per interval for the 1 to 18 day period, the original sample size was greatly reduced. The final number of animals used is indicated in each of the graphs. The proportion of labeled to unlabeled cells was determined for each animal in all age groups on samples of 1000 to 3000 cells in each of two regions, the renal corpuscle, and the remaining part of the nephron tubule. In the newborn kidney, counts were also made in the nephrogenic zone. No distinction was made during counting between the glomerulus and its capsule since the proportion of labeled cells appeared to be equal in both parts of the renal corpuscle. In the region of the tubules, labeled cells in the cortex were found to exceed those in the medulla by four to five times in the three younger age groups, but in the kidney of the 13 month mouse, labeled cells were only rarely encountered in tubules within the medulla. There have been other reports of the relative paucity of labeling and of mitotic activity of cells within the medulla [19, 251, but in comparisons of the mitotic activity of cells of the outer and inner parts of the medulla in rats up to 2 months of age, the outer medulla almost equals the cortex in mitotic activity [24]. In the present study it was decided to randomize counts for the nephron tubules to include cells situated in the cortex and all of the medulla. In the nephrogenic zone, counts were randomized to include cells comprising its entire depth. The dilution of the label over an 18 day period was determined from mean grain counts obtained from samples of 50 labeled cells in each of the regions noted above. The individual grain counts per animal were also used in a few instances for the construction of histograms. The method of grain counting described by Kisieleski et al. [ll] was used. Only in the newborn was it necessary to compare grain counts across all cell types. These comparisons‘were facilitated by a similarity in the mean grain counts of cells of all three zones at 1 day following the uptake of the labeled thymidine. Experimental
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RESULTS
Labeling of cells of the renal corpuscle and the nephron tubule at 1 hr following the administration of 3H-thymidine in the newborn, 2, 4 and 13 month old mouse.-In Fig. 1 the number of labeled cells per thousand in the renal cor-
2
4
b
8
10
12
14
Age in months
Fig. l-Number of labeled cells per thousand in two regions of the mouse kidney at 1 hr following *H-thymidine injection, plotted against age. Each point represents mean counts from the following numbers of animals: two animals at the newborn interval, 6 animals at the 2- and at the 4month intervals, and 12 animals at the 13-month interval. The vertical bar (I) indicates *one standard deviation, l - l , cells of renal corpuscle; 0 - - 0, cells of nephron tubule.
puscle and tubule at 1 hr after the injection of labeled thymidine is plotted against age. At birth there is no difference between these regions in their incidence of labeling. Both regions show a sharp decline in labeling from birth to 2 months, and a continued but very slow decline from 2 to 13 months. The overall decline is of the same order for both regions, but at the 13-month interval, a 1 to 2 difference in labeling appears. When the standard deviations of the means at the 13-month interval (see Fig. 1) are used to assess this large difference, it is found to be statistically insignificant. Thus, in the kidney of the 13-month mouse, the number of cells in DNA synthesis is of the same order for the whole of the nephron tubule and for the whole of the renal corpuscle. And from birth to 13 months there appears to be no change in this relationship. Labeling of cells of the renal corpuscle and the nephron tubule from 1 to 18 days following aH-thymidine administration in the 2-, C- and 13-month-old Experimental
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mouse.-Approximately one specimen per age group per daily interval was retained for these observations. The percentage of labeled cells was found to fluctuate randomly within the 1 to 18 day period for all three age groups. The mean grain counts showed a similarly random fluctuation for this period. The absence of either an increase in labeled cells or of a decrease in h
240.5
Fig. 2.-Number of labeled cells per thousand in three regions of the kidney of the newborn mouse, from 1 to 16 days following a single injection of SH-thymidine. Each point represents a single animal. A, nephrogenic zone; o-0, renal corpuscle; 0 - - 0, nephron tubule.
the mean grain count of the labeled cells suggests that in these two regions of the 2- to 13-month mouse kidney the average time between two successive mitotic divisions is longer than 18 days. Labeling of cells in the nephrogenic zone, the renal corpuscle and the nephron tubule from 1 hr to 16 days following 3H-thymidine administration in the newborn mouse.-Changes in the proportion of labeled cells in three regions of the newborn kidney were observed between 1 hr and 16 days following a single injection of labeled thymidine (Fig. 2). Counts for the nephrogenic zone terminate at 7 days since this zone is no longer present after this time. From birth to 7 days new renal corpuscles and nephron tubules continue to be formed from cells of this zone by differentiation and migration [7, 83. In the present series, the nephrogenic zone at birth has a depth of approximately 70 cells and forms a considerable part of the kidney. These cells are arranged into tightly packed whorls which cap and occasionally surround the adjacent renal corpuscles and tubules. Between 1 hour and 1 day this zone undergoes a reduction in cell depth by approximately oneExperimental
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half. Reduction in depth and in compactness continue to the 7th day when the zone becomes visible only as discontinuous masses of cells of varying depth and arrangement. At the time of initial labeling, 1 hr post-injection, approximately 1 of 4 cells of the nephrogenic zone is labeled whereas a relatively small number, 1
Fig. S.-Mean grain counts in three regions of the kidney of the newborn mouse, from 1 to 16 days following a single injection of aH-thymidine. Each point represents a single animal. A-A, nephrogenic zone O-O, renal corpuscle, o - - 0, nephron tubule.
of 25, is labeled in the renal corpuscles and tubules. In view of these initial differences and the known fate of cells of the nephrogenic zone, it is to be expected that the counts of labeled cells of the renal corpuscle and tubule will rise continuously to the 7th day since for every four cells that they incorporate approximately one will be labeled. On the other hand, as the nephrogenic zone loses cells, no change in its proportion of labeled cells should occur, if cell loss occurs on a random basis. For this reason the points representing the daily counts for the nephrogenic zone were not connected in Fig. 2. Changes in the proportion of labeled cells between 1 hr and 16 days following thymidine administration include the following. In the nephrogenic zone there appears to be only random fluctuation during the first 3 days but a distinct lowering occurs between 4 and 7 days. The renal corpuscles and tubules show continuous rises in the proportion of labeled cells between 1 hr and 7 days. The increase between 1 hour and 1 day reflects largely the division of cells which are in DNA synthesis at the time of thymidine administration. The increase between 1 and 7 days will reflect acquisition of cells from the nephrogenic zone but it could also reflect, at the terminal part of the curve, a new division of labeled cells of the renal corpuscle and tubules Experimental
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present in these regions at birth or acquired sometime thereafter. Between 7 and 8 days both regions show an abrupt decline in labeled cells and this is followed by a second increase at lo-11 days which is maintained till the 16th day. The mean grain counts in the newborn (Fig. 3) were used to (1) determine
2
15 10 15 I
10
1
2 days
51J41LmLL 10
20
30
10
20
30
1 day 10 15 1
Fig. 4.-Number Each histogram days following
I
of grains per nucleus in cells of two regions of the kidney of the newborn mouse. is based on counts of 50 nuclei per region per animal killed at 1, 2, 7, 11 and 16 a single injection of labeled thymidine.
the average length of the intermitotic interval of cells of the nephrogenic zone, the renal corpuscle and the nephron tubule; (2) the source of newly labeled cells found in the renal corpuscle and tubules between 1 and 7 days and between 10 and 16 days following labeling, when the first and the second rise in labeled cells occurred. Mean grain counts for the nephrogenic zone show an approximate halving over the 1 day counts between 2 and 3 days, indicating the occurrence at this time of a mitotic division subsequent to the initial division which followed Experimental
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thymidine uptake. Thus, for cells of the nephrogenic zone labeled at birth, the intermitotic interval which follows the initial division has an average length of I to 2 days. Labeled cells which are still present in the nephrogenic zone between the 4th and 7th day do not show an additional decline in grain number. In the renal corpuscle and tubules, the mean grain counts are not appreciably altered from the first to the 4th day, when these regions showed a marked rise in labeled cells. Grain halving in these labeled cells occurs gradually and is not completed until the 1.Ith day. From this it may be assumed that a division subsequent to the initial division occurs approximately on the 1 lth day. Thus, for cells of the renal corpuscle and tubules labeled at birth, the intermitotic interval has an average length of approximately 10 days. It EolLows from these comparisons of mean grain counts that between 1 and 7 days the source of newly labeled cells found in the renal corpuscle and tubules is the labeled cell of the nephrogenic zone. This is supported by a gradual fall in the mean grain count in these two regions coincident with their gradual acquisition of cells of lower grain count from the nephrogenic zone. It follows also that the increase in labeled cells in these two regions between 31 and 16 days is to be attributed to a division of cells present in these regions at birth and labeled at the time of thymidine uptake. Histograms of the individual grain counts per specimen were also constructed (Fig. 4) to explore the possibility that cells of halved grain count acquired from the nephrogenic zone had, perhaps, undergone a second division, with a resulting second hiving of their grains and a consequent lowering of the mean grain counts of both the renal corpuscle and nephron tubule. The histograms do not support the possibility that any given segment of the population of labeled cells is exclusively involved in mitotic activity at 11-16 days post-injection. When the 11 and 16 day histograms are compared with the 1 and 2 day histograms, rather than finding a widening of the range, one finds an overall shift toward a reduced number of grains per cell, indicating that all cells are contributing to a lowering of the mean grain counts. A histogram of the individual counts at 7 days was included to demonstrate the widening of the range and the increase in incidence of cells of lower grain count which predictably follow the acquisition of cells of lowered grain count from the nephrogenic zone. The grain counts could be used only indirectly to account for the loss of labeled cells in the nephrogenic zone between 4 and 7 days and in the renal corpuscles and tubules between 7 and 8 days. In the first instance the loss at 4 days follows a mitotic division which results in a reduction of the mean Experimenlal
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grain count to six grains. With the ensuing loss of intensity of label per’nucleus, an artifactual loss of labeled cells may easily occur at the lower end of the range. The second instance of cell decline is of disturbingly large magnitude and possibly reflects a number of factors of which the most discernible include the following. With the artifactual loss of labeled nephrogenic cells, only 1 of 8 cells acquired at 7 days is a labeled cell. Sometime between 7 and 8 days the nephrogenic zone will be lost entirely as a source of cells whereas the unlabeled cells present in the renal corpuscle and tubule will continue to divide resulting in a decrease in the proportion of labeled cells. This decrease will continue until such time as the labeled cells are prepared to divide again. This doesn’t occur until the 1 lth day. Undue variation resulting from smallness of sample size at this critical time may also be a factor.
DISCUSSION
Studies of organ growth have been vastly broadened in scope by the introduction of a method for the labeling of cells and their subsequent identitication. Some of the many features of this method have been utilized in the present study of the mouse kidney in observing changes in the proliferative activity of several regions of this organ and in the proliferative activity of the labeled cells of these same regions over a relatively short period of time. Inasmuch as the percentage of cells labeled by 3H-thymidine has been shown to exceed the percentage of cells simultaneously observed in mitosis by 5 to 15 times [19, 261, the labeling method provides data on the proliferative activity of an organ of a larger order than can be obtained by the method of mitotic counts. This feature is especially useful in determining the proliferative activity of organs such as kidney in which mitotic activity decelerates so markedly with age that it is frequently thought to be absent. The additional feature of grain halving during successive divisions of labeled nuclei [2. 5, 9, 10, 28, 301 further provides data for estimating the average length of the intermitotic interval of labeled cells. In organs having a relatively slow growth this interval would otherwise be determinable only indirectly, from a consideration of the mitotic index and population size. In so far as the label remains, the method also provides a marker for migrating cells, as demonstrated in epiphyseal plate cartilage and periosteum [10, 291, in intestinal and esophageal epithelium [2, 16, 18, 20, 221 and in hemopoietic tissue (see [27]). In the present study this feature permitted observations of the rate at which the undifferentiated cells of the kidney of the newborn mouse differentiate and migrate to form renal corpuscles and nephron tubules. Experimental
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All age groups: proliferative activity in the renal corpuscle and nephron tubule.-The incidence of labeling at 1 hr following the administration of 3H-thymidine was found to decline with age for the renal corpuscle and nephron tubule. In the newborn a proliferation rate of 40 cells per thousand was obtained for each of these regions. From birth to 2 months these rates decline sharply to approximately three cells per thousand. Beyond 2 months a decline of much smaller magnitude occurs providing rates of approximately two cells per thousand at 4 months, and approximately one cell per thousand at 13 months. The magnitude of change among these older age groups approaches that found for periosteal cells of mice of similar ages [29]. When the frequencies of labeling in the renal corpuscle and tubules are plotted against age, one obtains for each region a curve that is inversely related to the cumulative growth curve of the kidney [14]. Thus one finds that, at 2 months, coincident with the beginning of the decelerated phase of growth of the whole body [23] as well as of the kidney, cell proliferation in two regions of the kidney has also entered a decelerated phase. Within the context of the present study it cannot be determined whether proliferative activity within the renal corpuscle and nephron tubule between 2 and 13 months will contribute to their own growth in volume, or whether this activity represents only population renewal by the numerical replacement of dying cells. It is the additive aspect of cell proliferation which tends to be emphasized when one considers that there is continued growth of the whole organ, which in itself reflects the continued growth of the glomeruli, the non-glomerular cortex and the medulla, as shown from studies of the kidney of the rat and man [1, 121. From findings that continued growth from early maturity into middle and old age of the non-glomerular cortex and medulla do not reflect a hypertrophy of the cells of the nephron tubule [ 11, it follows that growth of these two regions containing the tubules represents, in the aging animal, an increase in tubule length, and that this increase must be dependent upon the addition of new cells. For the glomeruli of the aging animal, increase in cell volume cannot be ruled out as a factor in glomerular growth [l] until data are obtained to indicate otherwise. But with the finding of continued proliferation of the renal corpuscle in the 2- to 13-month-old mouse, there is no need for minimizing the contribution of cell proliferation to the growth of the glomerulus of the aging animal. All age groups: length of the intermitotic interval of cells of three regions of the mouse kidney.-For the newborn, changes in the frequency of labeled cells and in the mean and individual grain counts were observed from 1 to 16 days following a single injection of 3H-thymidine. Cells of the renal corExperimental
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puscle and nephron tubule which are labeled at birth were found to have an intermitotic interval of approximately 10 days. For the labeled undifferentiated cell of the nephrogenic zone, the length of the intermitotic interval is approximately l-2 days. The subsequent mitotic interval of cells which remain in the nephrogenic zone is either of the same or of longer duration but could not be determined in the present series due, perhaps, to too great a dilution of the label. Intermitotic intervals for cells of other organs of the newborn have not been reported, although intervals of 14, 2 and 5 days have been reported for mesenchymal cells in three regions of the tibia of the g-dayold rat [30], and an interval of l& days has been reported for epithelial cells established in vitro from fetal calf liver [ 151. For the 2-, 4- and 13-month-old mouse, changes in the frequency of labeled cells and in the mean grain count were observed from 1 to 18 days following a single injection of 3H-thymidine. The counts remained randomly distributed during this interval, suggesting an intermitotic interval with a duration longer than 18 days. An intermitotic interval of 30-50 days, based on data relating to the mitotic index, has been reported for cells of the glomerulus and tubules of the kidney of the adult rat and mouse [26]. Further investigation will establish whether differences in this interval exist among the age groups used in the present study. The newborn: changes in the proportion of labeled cells in three regions of the kidney at 1 hr and from 1 to 16 days following a single injection of labeled thymidine.-This 16-day period is a period of rapid growth. Reportedly there is more than a doubling in size of the mouse kidney from birth to 2 weeks of age [4], and possibly the magnitude of this increase, if quantified, would approach that of the rat kidney where the volume doubles from birth to 3 days and doubles again from 3 to 10 days [ 11. At 1 hr following thymidine administration, 240 cells per thousand were found labeled in the nephrogenic zone. This exceeds by six times the number of cells labeled in the renal corpuscle and nephron tubule, where approximately 40 cells per thousand were found. Similar differences in the mitotic activity of these regions have been reported for the kidney of the rat taken just prior to term [25]. Cells of the very actively proliferating nephrogenic zone will contribute to the formation of new renal corpuscles and nephron tubules until the zone is exhausted of cells [S]. Dissolution of this zone occurs at 7-8 days in the present series. In the rat this occurs at lo-12 days [3] and at 10 days in the human [6]. It would thus appear that during the first postnatal week the nephrogenic zone provides the kidney with a source of cells far out of proportion to its own relative volume. Judging only from the extent to which labeled cells differenExperimental
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tiate between 1 hr and 3 days and again from 3 to 7 days it appears that a maximal contribution to kidney growth is made during the earlier period. From the rapid reduction in the depth of this zone at 1 day, the maximal contribution would appear to be made within the first day. However, this reduction in depth is accompanied by its circumferential growth as it keeps pace with the rapidly enlarging regions within. Changes in the number of labeled cells from 1 to 16 days following thymidine administration were otherwise observed primarily to corroborate the time of a second mitotic division of cells labeled at 1 hr following thymidine administration. In following these changes two periods of accumulation of labeled cells were found in the renal corpuscles and tubules. The first of these periods occurs from 1 to 7 days following thymidine administration. On the basis of grain counts this accumulation of labeled cells was attributed to the incorporation of labeled, newly formed renal corpuscles and tubules derived from the undifferentiated cells of the nephrogenic zone. In view of the fact that the renal corpuscles and tubules remain equally labeled from 1 to 7 days and that the renal corpuscles comprise only a small part of the kidney [ 121, it follows that the nephrogenic zone contributes numerically more cells to tubule formation than to the formation of renal corpuscles. It would thus appear that morphogenesis of the tubules at this time involves to a lesser extent the growth of the renal vesicle between its two poles [8] and to a greater extent a significantly large splitting away of presumptive renal vesicle cells. The second period of labeled cell accumulation occurs from 10 to 16 days following thymidine administration and follows, by several days, dissolution of the nephrogenic zone. On this basis and from a consideration of changes in the grain counts, this second accumulation of labeled cells was considered to be a consequence of a second division of renal corpuscle and tubule cells labeled at birth or of labeled cells acquired by these regions shortly after birth. CONCLUSIONS
Age related changes have been found in the proliferative activity of the renal corpuscle and nephron tubule regions and also in the length of the intermitotic interval of cells within these regions. From the newborn to the older age groups there is a decrease in proliferative activity of the order of 40 : 3 : 2 : 1, and there is an increase in the length of the intermitotic interval of undetermined magnitude. When the age factor is kept constant, no differences are found between these two regions. However, in the newborn, when these two regions are compared with a third region, the nephrogenic zone, large Experimental
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differences appear. The nephrogenic zone has a proliferative activity which exceeds that of the other two regions by six times, whereas the length of the intermitotic interval of the already differentiated cells exceeds that of the nephrogenic zone by approximately five to six times. In other studies dealing with variation with age in these two aspects of growth, a lengthening of the generation cycle by approximately one fifth has been found in intestinal epithelium [17], but age differences in the proliferative activity of this epithelium at zero time have not been found [16, IS]. The age groups used in these instances range from 3 to 31 months, overlapping the older age groups used in the present study and in a study of the proliferation of periosteal cells during aging [29]. In all these organs or tissues of older animals, the differences in proliferative activity tend to be of a small and possibly insignificant order. This suggests that the relationship between the number of cells found proliferating and the length of the intermitotic interval of these cells will tend not to be sharply defined in organs of older animals. In younger age groups in which regional variation in these two aspects of growth have been examined, differences have been reported for both the proliferative activity and length of the intermitotic interval in epiphyseal cartilage [lo] and mesenchymal cells [30] of the tibia. In the present study, in the two instances in which large differences in proliferative activity appear, where the kidney of the very young and old are compared, and where different regions of the newborn kidney are compared, it is significant that one finds an inverse relationship between the proliferative activity of the organ or region and the length of the intermitotic interval of its dividing cells. When one examines data given in the regional studies of cartilage and bone in the studies cited above, one finds that there is supporting evidence for this relationship.
SUMMARY
In the kidney of newborn, 2-, 4- and 13-month-old mice, the number of cells per thousand labeled after a single injection of 3H-thymidine declines with age. This decline is of the order of 40: 3:2: 1 for the cells of each of two regions, the renal corpuscle and the nephron tubule. Labeling of the nephrogenic zone of the newborn, which contributes cells for additional formation of the renal corpuscles and nephron tubules, was found to exceed by six times the labeling of each of the already differentiated regions. Intermitotic intervals were determined from grain counts recorded from Experimental
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0 to 18 days following a single injection of SH-thymidine; at 2, 4 and 13 months, this interval is longer than 18 days for cells of the renal corpuscle and nephron tubule; in the newborn it is ten days for these same cells and approximately 2 days for cells of the nephrogenic zone. Changes with age in proliferative activity are discussed with respect to known age changes in kidney volume. Regional differences in proliferative activity of the newborn kidney are discussed relative to the duration of the intermitotic interval of the cells of these regions and relative to the rate of differentiation of cells of the nephrogenic zone. REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
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