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A Study of Induced Renal Hyperplasia using Autoradiography
Tn~ .JouRI\~Ati OF U:rwLOG\T Vol. \J;3, No. 1 January 1065 Copyright © 1065 by The "VVilliams & \Yilkins Co. Printed in (I.S.ll
A STUDY OF INDFCED RE1'AL HYPERPLASIA USING AUTORADIOGRAPHY RCSSEL J HEITER*
AND
CHARLES E. 1\/lcCREfGHT
From !he Depmlm.ent of A natmny, FJowrnan Gray School of
lt has been shown that kidney hypertrophy in rodents can be induced by any of several experiJnental procedures. Interest in histologic and eytologic: changes within such enlarging kidneys has increased within recent years. Se\·eral investigators have observed that .~urgical removal of a single kidney 1esults in a significant increase in mitotic activity in the tubules of the contralateral organ the second or third postoperative day. 1 -3 lVIcCreight and Sulkin have demonstrated that the proportion of the total kidney mass removed determines the degree of the proliferative response in the remaining kidney complement. 4 -Following bilateral heminephrec:tomy they found a 6 to 7-fold increa~e in mitotic activity in the proximal convoluted tubules of the remaining kidney hah-es. After increasing the amount of surgical deletion to seven-eights of the kidney complement, a 25-folcl increase in mitotic counts in the proximal convoluted tubules of the remaining unilateral kidney fourth.~ was observed. All previous studies were performed with routine or specialized methods of ~taining and cytological observation. lt was thought desirable to investigate further the problem of compensatory rnual hyperplasia following unilateral nephrcctomy with the technique of autoracliog raphy, using tritiated-thymidinc. This would also be important to establish a base linr; for further
Winston-Salem, North Carolirw
investigations of compensatory renal hyperplasia. using autora.diography. Thymidine is a specific precursor for desoxyribonuc:leic acid Although synthesis of DNA precedes mitosis in the rat kidney by approximately 12 hours, the incorporation of the radioactive cornpound into newly-forming nuclei over a short measured interval would prnvide an indirect, but reliable method of determining mitotic rate in tissues, MA'rEHJALS AND METHODS
Twelve male albino rats of the Spragnt'-Dawley strain, 3 to 4 months of age, were used in thi8 experiment. The animals were kept three in a cage and food and water were supplied ad libitum. To conform with previous experiments, 3 · operations were performed with etl1er anesthesia and a single kidney was removed surgically an intra-abdominal approach from six of the 12 arnmals between 2: 00 and 4: 00 p.m. The remaining rats served as controls, On the' second postoperative day at 10: 00 a.m., a single intxapNitoneal injection of aqueous tritiatrd-thymidine (specific activity, 1 .9 C /rn!\1) was given to each of the control and experimental animals. The was one microcurie per gram of body weight_ On the Rame day at .2: 00 p.m., 48 ho urn following unilateral nephrectomy of the experirnental rats, all animals were sacrificed by over-exposure to ether_ Remaining kidneys from experimental animal., and both kidneys from control animals 1vere excised, sliced, and fixed in JO per ce1H neutral formalin. Following paraffin embedding, sections were cut at 5 µ. Kodak AR-10 stripping; film was applied tu the .,ec:tions, a.ncl the exposure time wa.s 32 days. Preparations 1verc developed, fixed, and stained with Harris' hexatoxylin and eosin. To determine the mean mimber of tubular cells per high power field ( X400), counts of tubu Jar cells (those of vessels and connective tissue were excluded) were made in .25 fields each of cortex a.ncl rneclulla. In addition, counts of labeled tubular cells in .50 random fields each of
Accepted for publication June 23, 1964. This investigation was supported by a grant H-·G8GO from the N'ationa.l Heart Institute, National Institutes of Health. The United States Public lfoalt.h Serviee. Present address: Physiology Division, Directo-
rate of 1\.fedical .Research, EJgewood Arsenal, .:VIaryland. . . 1 Sulkin, N. M .. Cytologic studies of the remaming kidney follo,Ying unilateral nephrectomy in the rnt. Ana1,. Hee., 105: !JS--112, Hl49. ' Rollason, H. D.: Compensatory hypertrophy of the kidney of the young ra.t with special em-
plrnsis on the role of cellular hyperplasia. Anat, Rc,c , 104: 263-28.5, Hl49. 3 McCreight,. C. E. and Sulkin N M.: Cellular proliferatioi1 in the kidneys of young and senile rats following unilateral nr,phrectomy. J. Geront., 14: 440-443, 19.59, 1 McCreighL C. E. and SuJkin, N, M.: Com--
pensatory rcrial hyperplasia following experimental surgical deletions of the kidney comple-
ment. Amer, .L Anat., 110: 19D-202, 19(;2, 27
28
REITER AND MCCREIGHT
OBSERVATIONS
the remaining kidneys was 0.64 per cent of the population of tubular cells. Therefore, the percentage of labeled cells in the medulla of remaining kidneys was 12 to 13 times that in the medulla of control kidneys. The labeled cells were found in renal tubules of all types. However, the cells which had incorporated the tritiated-thymidine were not uniformly dispersed throughout the renal parenchyma. A greater percentage of the cells in some areas than in others showed synthesis of DNA. Occasionally, in both control and remaining kidneys, a labeled cell was found in a renal c01vuscle, but rarely had cells of any type other than tubular cells taken up the labeled compound.
Statistical analysis indicated that the mean number of cells per field of either cortex or medulla did not differ significantly for control and experimental kidneys. However, there was a much greater density of tubular cells within the medulla than in the cortex of the rat kidney. The cortical areas had a mean of 437 (standard error (S.E.) ± 5.06) cells per field, while in the medulla the mean was 732 (S.E. ± 7.42) cells per field. This is a ratio of 1.7 medullary cells to each cortical cell. The incidence of labeled cells in the cortex of control kidneys was very low. A range of O to 3 such cells per field was found in the 50 areas considered, with a mean of 0.62 labeled cells per field. Two days following unilateral nephrectomy the mean number per field had increased to 3.32 labeled cells (table 1). Further analyses of the data indicated that 0.14 per cent of the cortical cells of the control kidneys had taken up the tritiated-thymidine, compared to 0.76 per cent of the cells of the remaining kidneys. These figures indicated a 5 to 6-fold increase in labeled cells in cortical tissue of remaining kidneys over those of controls. The mean number of labeled cells per field in the medulla of the control kidneys was 0.36, about one-half that in the cortex of the control kidneys. However, in the kidneys remaining after unilateral nephrectomy, the mean number of labeled cells per field in the medulla had increased to 4.72, an even greater number than that recorded per field for the cortex of remaining kidneys (table 1). Since there is a greater density of cells in the medulla, the percentage of labeled cells in this region of the control kidneys proved to be only 0.05 per cent, while that in the medulla of
Sulkin1 reported that counting of 100 oil immersion fields in each of cortex and medulla of the kidney of the rat revealed that there were 45 cells per field in the former and 81 cells per field in the latter. The results obtained in this investigation, with the ratio 1: 1.7 of cortical to medullary cells, are in close agreement with the findings of this investigator. The work of McCreight and Sulkin3 • 4 showed that a 6 to 7-fold increase in the mitotic activity of the proximal convoluted tubules occurred 48 hours after surgical deletion of one-half of the renal tissue mass of the rat. Similar results were obtained if a single kidney were removed or if bilateral heminephrectomies were performed. The findings of the present work showed that the labeling index of the cortex had increased 5 to 6-fold on the second postoperative day following unilateral nephrectomy. This increase is slightly lower than that observed by McCreight and and Sulkin. The lower results could be explained as follows. Blumenfeld 5 has shown that the normal mitotic activity of the rat kidney is highest between 2:00 and 4:00 p.m. Since the animals used in this investigation were sacrificed during this period, the mitotic rate should have been highest at the time of sacrifice. However, as stated previously, synthesis of nucleoprotein in the rat kidney precedes mitosis by approximately 12 hours. Had the animals been sacrificed during the early morning hours of the same day, the labeling index of the cortex of the remaining kidney might well have been approximately 6 to 7 times as great as that of the control kidney. 5 Blumenfeld, C. M.: Periodic and rhythmic mitotic activity in the kidney of the albino rat. Anat. Rec., 72: 435-443, 1938.
1. Comparison of mean numbers (± standard error) of cells labeled with tritiated-thymidine in control kidneys of rats and in remaining kidneys following unilateral nephrectom71
TABLE
Region
Cortex Medulla
No. of Fields per Kidney
50 50
Con. Kid., Labeled Cells per HP Field
Rem. Kid., Labeled Cells per HP Field
P Value
--0.62 ± 0.11 3.32 ± 0.24 <0.001 0.36 ± 0.08 4. 72 ± 0.45 <0.001
cortex and medulla for control animals and similar observations for experimental animals were made.
I
r
DISCUSSION
29
INDT]CED RENAL HYPERPLASIA
With regard to tlw of labeled cells in the medulla of eontrol .,pecimens, Enesco has stated that an extremely low mitotic rate is observed also in the medulla of the normal mouse kidney." In the present a 12 to 13-fold increase in the labeling irnlex of the medulla was recorded 48 hours after ,,urgieal removal of a single kidney. However, ~inc<, the .number of medullary cells taking np tritiated-thymidine in controls was rC'latively low, the percentage of labeled cells in the rnecfolla after unilateral nephrectomy still did not reach the percentage of labelc:d. cdls 1n the cmtex of the, sarne kidneys. These r0~ults corroborate tlw of Rolla-· :,on,' who obsern;cl a gr0ater number of cortical than of meclullary cells in rnito~i.s on the second day after unilateral nephrectomy. That the labeled cells me not distributed cvrnly throughout the n'rml parenchyma is not "Enesco, lVL · PersoIJaJ Cl1mmunica.tinn,
a new finding. Sulkin 1 olisc1Ted that unilateral nephrectorny in the rnt some areas of the remaining kidney were active rnitohc:ally, while others were relatively inaGti1·t,. No '"'''"U'"-"' tion has been ad,·anced as yet for this non. SUMMARY
To establish n ha'ic line for future autorndro graphic: investigations, cornpensatory reual p]a;;ia was studied tL'iing Labeled cdb wen, infrequent in the tulrnlar tissues of control kidne,rn. In the kidneys remain, ing after unilateral nephreetomy there wa.s a ;S fi-folcl increase in the cmtcx and a 12 l:o 13.fold increa;;c in the medulla of Jabelrd tubular ccll.s over the nurnbern ~een in control Liiiwes. Th(, findings obtained here arc in agreement with the obsern1tions made in earlim innistigations with. routine mEthods of staining and mitotic ob~erva. tion,
A STUDY OF INDFCED RE1'AL HYPERPLASIA USING AUTORADIOGRAPHY RCSSEL J HEITER*
AND
CHARLES E. 1\/lcCREfGHT
From !he Depmlm.ent of A natmny, FJowrnan Gray School of
lt has been shown that kidney hypertrophy in rodents can be induced by any of several experiJnental procedures. Interest in histologic and eytologic: changes within such enlarging kidneys has increased within recent years. Se\·eral investigators have observed that .~urgical removal of a single kidney 1esults in a significant increase in mitotic activity in the tubules of the contralateral organ the second or third postoperative day. 1 -3 lVIcCreight and Sulkin have demonstrated that the proportion of the total kidney mass removed determines the degree of the proliferative response in the remaining kidney complement. 4 -Following bilateral heminephrec:tomy they found a 6 to 7-fold increa~e in mitotic activity in the proximal convoluted tubules of the remaining kidney hah-es. After increasing the amount of surgical deletion to seven-eights of the kidney complement, a 25-folcl increase in mitotic counts in the proximal convoluted tubules of the remaining unilateral kidney fourth.~ was observed. All previous studies were performed with routine or specialized methods of ~taining and cytological observation. lt was thought desirable to investigate further the problem of compensatory rnual hyperplasia following unilateral nephrcctomy with the technique of autoracliog raphy, using tritiated-thymidinc. This would also be important to establish a base linr; for further
Winston-Salem, North Carolirw
investigations of compensatory renal hyperplasia. using autora.diography. Thymidine is a specific precursor for desoxyribonuc:leic acid Although synthesis of DNA precedes mitosis in the rat kidney by approximately 12 hours, the incorporation of the radioactive cornpound into newly-forming nuclei over a short measured interval would prnvide an indirect, but reliable method of determining mitotic rate in tissues, MA'rEHJALS AND METHODS
Twelve male albino rats of the Spragnt'-Dawley strain, 3 to 4 months of age, were used in thi8 experiment. The animals were kept three in a cage and food and water were supplied ad libitum. To conform with previous experiments, 3 · operations were performed with etl1er anesthesia and a single kidney was removed surgically an intra-abdominal approach from six of the 12 arnmals between 2: 00 and 4: 00 p.m. The remaining rats served as controls, On the' second postoperative day at 10: 00 a.m., a single intxapNitoneal injection of aqueous tritiatrd-thymidine (specific activity, 1 .9 C /rn!\1) was given to each of the control and experimental animals. The was one microcurie per gram of body weight_ On the Rame day at .2: 00 p.m., 48 ho urn following unilateral nephrectomy of the experirnental rats, all animals were sacrificed by over-exposure to ether_ Remaining kidneys from experimental animal., and both kidneys from control animals 1vere excised, sliced, and fixed in JO per ce1H neutral formalin. Following paraffin embedding, sections were cut at 5 µ. Kodak AR-10 stripping; film was applied tu the .,ec:tions, a.ncl the exposure time wa.s 32 days. Preparations 1verc developed, fixed, and stained with Harris' hexatoxylin and eosin. To determine the mean mimber of tubular cells per high power field ( X400), counts of tubu Jar cells (those of vessels and connective tissue were excluded) were made in .25 fields each of cortex a.ncl rneclulla. In addition, counts of labeled tubular cells in .50 random fields each of
Accepted for publication June 23, 1964. This investigation was supported by a grant H-·G8GO from the N'ationa.l Heart Institute, National Institutes of Health. The United States Public lfoalt.h Serviee. Present address: Physiology Division, Directo-
rate of 1\.fedical .Research, EJgewood Arsenal, .:VIaryland. . . 1 Sulkin, N. M .. Cytologic studies of the remaming kidney follo,Ying unilateral nephrectomy in the rnt. Ana1,. Hee., 105: !JS--112, Hl49. ' Rollason, H. D.: Compensatory hypertrophy of the kidney of the young ra.t with special em-
plrnsis on the role of cellular hyperplasia. Anat, Rc,c , 104: 263-28.5, Hl49. 3 McCreight,. C. E. and Sulkin N M.: Cellular proliferatioi1 in the kidneys of young and senile rats following unilateral nr,phrectomy. J. Geront., 14: 440-443, 19.59, 1 McCreighL C. E. and SuJkin, N, M.: Com--
pensatory rcrial hyperplasia following experimental surgical deletions of the kidney comple-
ment. Amer, .L Anat., 110: 19D-202, 19(;2, 27
28
REITER AND MCCREIGHT
OBSERVATIONS
the remaining kidneys was 0.64 per cent of the population of tubular cells. Therefore, the percentage of labeled cells in the medulla of remaining kidneys was 12 to 13 times that in the medulla of control kidneys. The labeled cells were found in renal tubules of all types. However, the cells which had incorporated the tritiated-thymidine were not uniformly dispersed throughout the renal parenchyma. A greater percentage of the cells in some areas than in others showed synthesis of DNA. Occasionally, in both control and remaining kidneys, a labeled cell was found in a renal c01vuscle, but rarely had cells of any type other than tubular cells taken up the labeled compound.
Statistical analysis indicated that the mean number of cells per field of either cortex or medulla did not differ significantly for control and experimental kidneys. However, there was a much greater density of tubular cells within the medulla than in the cortex of the rat kidney. The cortical areas had a mean of 437 (standard error (S.E.) ± 5.06) cells per field, while in the medulla the mean was 732 (S.E. ± 7.42) cells per field. This is a ratio of 1.7 medullary cells to each cortical cell. The incidence of labeled cells in the cortex of control kidneys was very low. A range of O to 3 such cells per field was found in the 50 areas considered, with a mean of 0.62 labeled cells per field. Two days following unilateral nephrectomy the mean number per field had increased to 3.32 labeled cells (table 1). Further analyses of the data indicated that 0.14 per cent of the cortical cells of the control kidneys had taken up the tritiated-thymidine, compared to 0.76 per cent of the cells of the remaining kidneys. These figures indicated a 5 to 6-fold increase in labeled cells in cortical tissue of remaining kidneys over those of controls. The mean number of labeled cells per field in the medulla of the control kidneys was 0.36, about one-half that in the cortex of the control kidneys. However, in the kidneys remaining after unilateral nephrectomy, the mean number of labeled cells per field in the medulla had increased to 4.72, an even greater number than that recorded per field for the cortex of remaining kidneys (table 1). Since there is a greater density of cells in the medulla, the percentage of labeled cells in this region of the control kidneys proved to be only 0.05 per cent, while that in the medulla of
Sulkin1 reported that counting of 100 oil immersion fields in each of cortex and medulla of the kidney of the rat revealed that there were 45 cells per field in the former and 81 cells per field in the latter. The results obtained in this investigation, with the ratio 1: 1.7 of cortical to medullary cells, are in close agreement with the findings of this investigator. The work of McCreight and Sulkin3 • 4 showed that a 6 to 7-fold increase in the mitotic activity of the proximal convoluted tubules occurred 48 hours after surgical deletion of one-half of the renal tissue mass of the rat. Similar results were obtained if a single kidney were removed or if bilateral heminephrectomies were performed. The findings of the present work showed that the labeling index of the cortex had increased 5 to 6-fold on the second postoperative day following unilateral nephrectomy. This increase is slightly lower than that observed by McCreight and and Sulkin. The lower results could be explained as follows. Blumenfeld 5 has shown that the normal mitotic activity of the rat kidney is highest between 2:00 and 4:00 p.m. Since the animals used in this investigation were sacrificed during this period, the mitotic rate should have been highest at the time of sacrifice. However, as stated previously, synthesis of nucleoprotein in the rat kidney precedes mitosis by approximately 12 hours. Had the animals been sacrificed during the early morning hours of the same day, the labeling index of the cortex of the remaining kidney might well have been approximately 6 to 7 times as great as that of the control kidney. 5 Blumenfeld, C. M.: Periodic and rhythmic mitotic activity in the kidney of the albino rat. Anat. Rec., 72: 435-443, 1938.
1. Comparison of mean numbers (± standard error) of cells labeled with tritiated-thymidine in control kidneys of rats and in remaining kidneys following unilateral nephrectom71
TABLE
Region
Cortex Medulla
No. of Fields per Kidney
50 50
Con. Kid., Labeled Cells per HP Field
Rem. Kid., Labeled Cells per HP Field
P Value
--0.62 ± 0.11 3.32 ± 0.24 <0.001 0.36 ± 0.08 4. 72 ± 0.45 <0.001
cortex and medulla for control animals and similar observations for experimental animals were made.
I
r
DISCUSSION
29
INDT]CED RENAL HYPERPLASIA
With regard to tlw of labeled cells in the medulla of eontrol .,pecimens, Enesco has stated that an extremely low mitotic rate is observed also in the medulla of the normal mouse kidney." In the present a 12 to 13-fold increase in the labeling irnlex of the medulla was recorded 48 hours after ,,urgieal removal of a single kidney. However, ~inc<, the .number of medullary cells taking np tritiated-thymidine in controls was rC'latively low, the percentage of labeled cells in the rnecfolla after unilateral nephrectomy still did not reach the percentage of labelc:d. cdls 1n the cmtex of the, sarne kidneys. These r0~ults corroborate tlw of Rolla-· :,on,' who obsern;cl a gr0ater number of cortical than of meclullary cells in rnito~i.s on the second day after unilateral nephrectomy. That the labeled cells me not distributed cvrnly throughout the n'rml parenchyma is not "Enesco, lVL · PersoIJaJ Cl1mmunica.tinn,
a new finding. Sulkin 1 olisc1Ted that unilateral nephrectorny in the rnt some areas of the remaining kidney were active rnitohc:ally, while others were relatively inaGti1·t,. No '"'''"U'"-"' tion has been ad,·anced as yet for this non. SUMMARY
To establish n ha'ic line for future autorndro graphic: investigations, cornpensatory reual p]a;;ia was studied tL'iing Labeled cdb wen, infrequent in the tulrnlar tissues of control kidne,rn. In the kidneys remain, ing after unilateral nephreetomy there wa.s a ;S fi-folcl increase in the cmtcx and a 12 l:o 13.fold increa;;c in the medulla of Jabelrd tubular ccll.s over the nurnbern ~een in control Liiiwes. Th(, findings obtained here arc in agreement with the obsern1tions made in earlim innistigations with. routine mEthods of staining and mitotic ob~erva. tion,