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Contralateral Renal Hyperplasia and Increased Renal Function after Relief of Chronic Unilateral Ureteral Obstruction
0022-534 7/84/1325-1016$02.00/0 Vol. 132, November
THE JOURNAL OF UROLOGY
Copyright© 1984 by The Willia:11s & Wilkins Co.
Printed in U.S.A.
CONTRALATERAL RENAL HYPERPLASIA AND INCREASED RENAL FUNCTION AFTER RELIEF OF CHRONIC UNILATERAL URETERAL OBSTRUCTION WILLIAM N. CASTLE
W. SCOTT MCDOUGAL*
AND
From the Division of Urology, Department of Surgery, Dartmouth Medical School, Hanover, New Hampshire
ABSTRACT
Following relief of 1, 2 or 3 weeks of unilateral ureteral obstruction, contralateral compensatory renal growth and increased renal function were measured 3 and 6 months later. Compensatory growth occurred predominantly by hyperplasia, demonstrated by a significant increase in DNA content and a decrease in the RNA:DNA ratio (p <0.04). This is in contrast to compensatory growth following nephrectomy or unrelieved unilateral ureteral obstruction, which occurred primarily by hypertrophy with no significant change in DNA content but a significant increase in RNA content and the RNA:DNA ratio (p <0.04). Contralateral renal function in animals with relieved unilateral ureteral obstruction was greater than in controls with 2 normal kidneys (p <0.05). The contralateral increase in renal function was greater than that in animals subjected to ipsilateral nephrectomy or unrelieved ureteral obstruction, but this did not reach statistical significance. Thus, when growth occurred by hyperplasia, there was a trend to greater increases in renal function than when growth occurred by hypertrophy. Contralateral compensatory renal hyperplasia and increased renal function occurred in conjunction with a decrease in renal mass and function of the ipsilateral post-obstructed kidney. These experiments suggest that the post-obstructed, poorly functioning kidney stimulates contralateral hyperplastic growth and increased renal function. This hyperplastic response is different from the hypertrophic response following nephrectomy or unrelieved unilateral ureteral obstruction, implicating the post-obstructed kidney as the stimulus of the hyperplastic response. Compensatory renal growth and functional adaptation following chronic unrelieved unilateral ureteral obstruction has been shown to be similar to that following nephrectomy. 1 Growth occurs primarily by cellular hypertrophy with a subsequent increase in renal function. With relief of unilateral ureteral obstruction, the return of function in the post-obstructed kidney is inversely related to the length of time the kidney has been obstructed. 2 However, the compensatory growth response and its relationship to function after relief of obstruction have not been defined. There is evidence to suggest that humoral factors influence renal growth as stimulators or inhibitors of the compensatory response after unilateral nephrectomy or unrelieved ureteral obstruction. 3 These factors have not been identified nor has their site of origin been determined. The following experiments demonstrate that compensatory renal growth following relief of chronic unilateral ureteral obstruction occurs primarily by cellular hyperplasia. 4 This compensatory growth is associated with an increase in renal function. Moreover, the data suggest that the post-obstructed, damaged kidney is the stimulus for this unique response. MATERIALS AND METHODS
One hundred Long Evans male rats weighing between 125 and 150 grams were divided into 6 groups: sham controls, unrelieved unilateral ureteral obstruction, unilateral nephrectomy and unilateral ureteral obstruction relieved by microsurgical ureterovesical reimplantation at 1, 2 and 3 week intervals. All animals were anesthetized with pentobarbital, 50 mg./kg. body weight. Unilateral ureteral obstructions were created through Accepted for publication June 4, 1984. * Requests for reprints: Dept. of Urology, Vanderbilt University Medical Center North, Nashville, TN 37232. Read at annual meeting of the American Urological Association, New Orleans, Louisiana, May 10, 1984.
a midline incision by placing an occlusive ligature about the left ureter at the ureterovesical junction. Left nephrectomies were performed through a flank incision. Sham controls underwent a midline incision without manipulation of ureters. Rats undergoing relief of unilateral ureteral obstruction were divided into 3 subgroups: 1, 2 and 3-weeks' obstruction. At the end of the period of obstruction, the animals were anesthetized with pentobarbital (50 mg./kg. body weight), and the obstructed ureter was reimplanted into the bladder using 8-0 collagen (Ethicon) and an operating microscope. Control animals were not subjected to sham operations at this time. After all operations, the rats were placed in individual cages and allowed free access to rat chow and deionized water. All animals were allowed to recover for either 3 or 6 months, except for the 3-weeks' obstructed group which was studied only at 3 months. Following the 3 or 6 month recovery period, the rats were again anesthetized with pentobarbital, 50 mg./kg. body weight. They were placed on a thermoregulated table, and the carotid artery, jugular vein and both ureters (or 1 ureter and the bladder) were cannulated using PE-10 or PE-50 tubing. All reimplanted ureters were unobstructed; their diameters were greatly reduced as compared to their size at the time of reim plantation by approximately 80 to 90 per cent. An infusion of 10 per cent Inutest (Laevosan-Gesselschaft) and 0.5 per cent para-amino hippurate (PAH) (Merck, Sharp, and Dome) in lactated Ringer's solution was infused through the jugular vein at a rate of 10 ,ul./min./100 gm. body weight. Each rat also received a bolus of lactated Ringer's (equal to 2 per cent of his body weight) at the beginning of the infusion to replace evaporative losses during the procedure. The rats were allowed to equilibrate for 45 minutes with this infusion, followed by a 60minute collection of urine obtained under oil. At the midpoint and endpoint of the urine collection, a blood sample was obtained via the carotid artery. The experiment was terminated
1016
1017
CONTRALATERAL REt-;AL f-IYPERPLASIA
both and determining wet in pretins. One half of each kidney was prepared immediately determination by the modified Schmidt-Thannhauser for procedure,5 and for DNA determination by the diphenylamine reaction. 6 The other half of each kidney was placed in a preweighed tin, minced and dried at 37C for 3 days to determine
renal dry •Neight. Blood and urine were analyzed for inulin by the An throne Method, 7 P AH by the Bratton-Marshall reaction8 and osmolality by freezing point depression. Statistical analyses were performed using Student's t test. A level of p < 0.05 was assumed significant. RESULTS
L Renal dry weight (mg./100 gm. body weight) for individual kidneys measured at 3 and 6 months for control and experimental animals#
TABLE
Percent Change in Dry Weight Compared to Sham Operated Rats
Renal Dry Weight
Right Control Sham operated 3 months (no.= 10) 6 months (no. = 13) Left nephrectomy 3 months (no. = 8) 6 months (no. = 9) Left ureteral obstruction 3 months (no. = 7) 6 months (no. = 9) Experimental l Week obstruction/ relieved 3 months (no. = 8) 6 months (no. = 8) 2 Weeks obstruction/ relieved 3 months (no. = 11) 6 months (no. = 10) 3 Weeks obstruction/ relieved 3 months (no. = 6)
7.0 ± 0.6 6.7 ± 0.5
Left
Right
Left
6.6 ± 0.7 6.7 ± 0.6
10.3 ± 0.5* 9.8 ± 0.8*
+47 +46
9.3 ± 0.9* 9.1 ± 0.5*
+ +
+33 +36
8.2 ± 0.7** 7.2 ± 0.9
5.2 ± 1.1* 4.3 ± 1.1 *
+17 + 7
-36
10.2 ± 1.6* 9.2 ± 1.8*
3.9 ± 1.3* 3.0 ± 1.9
+46 +37
-41 -55
9.1 ± 0.7'''
2.7 ± 0.8*
+30
-59
-21
# Values are expressed as the mean ± SD. + No renal parenchyma. * Significantly different from sham controls (p <0.05). ** Significantly different from all controis (p <0.005).
TABLE 2.
Body weights at the time of clearance measurements were not significantly different for groups studied at 3 months. Sham operated controls were 493 ± 69 gm. (mean ± SD), 502 ± 49 gm. for left nephrectomy controls, and 504 ± 35 gm. for left unrelieved ureteral obstruction controls. Body weights for the experimental animals at 3 months were 502 ± 51 gm. for 1week obstructions, 495 ± 69 gm. for 2-weeks' obstructions and 514 ± 43 gm. for 3-weeks' obstructions. Body weights for the rats studied at 6 months demonstrated some variation. Sham operated controls weighed 537 ± 45 gm., left nephrectomy controls 637 ± 29 gm. and unrelieved left ureteral obstruction controls 603 ± 62 gm. The latter 2 groups are significantly different from sham operated controls (p <0.007). Body weights for the experimental animals at 6 months were 589 ± 57 gm. for I-week obstructions, which is significantly different from sham operated controls (p <0.02), and 525 ± 69 gm. for 2weeks' obstructions, which is not significantly different from the sham operated controls. Renal dry weights (table 1) demonstrated significant changes in renal mass. All groups, except the 1-week obstructed rats studied at 6 months, were significantly different from sham operated controls (p <0.005). The left nephrectomy and left unrelieved ureteral obstruction controls had significantly greater renal dry weights at 3 and 6 months (p <0.005). This is consistent with compensatory renal growth. There was no discernible renal parenchyma (histologically) in the left unrelieved ureteral obstructed kidney at 3 or 6 months. The increases in renal mass in the contralateral kidney of the nephrectomy and unrelieved ureteral obstruction controls were significantly greater (p <0.005) than those seen in the contralateral kidney in the 1-week obstructed rats at 3 and 6 months. However, there was no significant difference with longer periods of obstruction, at 2 and 3 weeks. The contralateral renal growth in animals whose obstruction was relieved at 2 and 3
RNA content, DNA content (mg./gm. renal weight), and RNA:DNA ratio for individual kidneys measured at 3 and 6 months for controls and after relief of obstruction for experimental animals# DNA Content
RNA Content Right Control Sham operated 3 months (no. 6 months (no.= Left nephrectomy 3 months (no. = 8) 6 months (no. = 9) Left ureteral obstruction 3 months (no. = 7) 6 months (no. = 9) Experimental 1 Week obstruction/ relieved 3 months (no. = 8) 6 months (no. = 8) 2 Weeks obstruction/ relieved 3 months (no. = 11) 6 months (no. = 10) 3 Weeks obstruction/ relieved 3 months (no. = 6)
40.40 ± 1.86 42.81 ± 2.90
Left
40.45 ± 3.37 42.86 ± 2.50
48.52 ± 7.12* 46.65 ± 6.46*
Right
22.75 ± 2.57 23.40 ± 3.29
RNA:DNA Left
22.19 ± 1.74 22.70 ± 2.91
21.86 ± 5.54 21.67 ± 3.45
42.82 ± 4.25 43.34 ± 2.89
+ +
20.76 ± 4.50 23.59 ± 1.66
Right
1. 794 ± 0.195
l.856 ± 0.240
Left
1.829 ± 0.180 1.916 ± 0.261
2.397 ± 0.803* 2.194 ± 0.455*
+ +
2.135 ± 0.363* 1.843 ± 0.148
+ +
42.1 7 ± 3.42** 41.03 ± 3.80**
42.69 ± 6.21 41.46 ± 4.16
27.02 ± 3.55**'' 25.58 ± 4.97**
28.05 ± 3.82* 31.81 ± 8.77*
1.653 ± 0.209** 1.682 ± 0.477
1.556 ± 0.241 * 1.401 ± 0.4 72*
34.65 ± 6.29*** 41.38 ± 3.45**
33.60 ± 6.48* 42.15 ± 4.35
26.34 ± 7.42*** 28.69 ± 5.89***
30.84 ± 5.93* 35.14 ± 7.15*
1.374 ± 0.300** 1.505 ± 0.346**
1.129 ± 0.307* 1.256 ± 0.322*
32.46 ± 4.28***
30.45 ± 1.62*
27.00 ± 3.73***
28. 75 ± 4.12*
1.327 ± 0.252**
1.051 ± 0.125*
# Values are expressed as the mean ± SD. + No renal parenchyma. * Significantly different from sham controls (p <0.05). ** Significantly different from control nephrectomies (p <0.03). *** Significantly different from all controls (p <0.005).
1018
CASTLE AND MCDOUGAL
TABLE 3.
Inulin, para-amino hippurate and osmolar clearances (µl./min./100 gm. body weight) for individual kidneys measured at 3 and 6 months for controls and after relief of obstruction for experimental animals# crnulin (GFR) Right
cosm
cPAH (ERPF)
(Osmolar Clearance)
Left
Right
Left
Right
285 ± 67 325 ± 97
856 ± 211 1075 ± 314
856 ± 211 1075 ± 314
10.36 ± 2.61 10.27 ± 2.63
Left
Control Sham operated 3 months (no. = 8) 6 months (no.= 8) Left nephrectomy 3 months (no. = 7) 6 months (no. = 7) Left ureteral obstruction 3 months (no. = 7) 6 months (no. = 6) Experimental 1 Week obstruction/ relieved 3 months (no. = 6) 6 months (no. = 7) 2 Weeks obstruction/ relieved 3 months (no. = 7) 6 months (no. = 9) 3 Weeks obstruction/ relieved 3 months (no. = 6)
285 ± 67 325 ± 97 303 ± 32* 359 ± 83
9.74 ± 2.01 10.27 ± 2.63
7.06 ± 2.47 14.67 ± 3.93
1164 ± 306* 1126 ± 377
311 ± 117 402 ± 92*
+ +
968 ± 255 1195 ± 322
+ +
9.59 ± 5.04 15.29 ± 4.88
+
309 ± 80 345 ± 145
159 ± 43* 204 ± 75*
1052 ± 349 960 ± 434
492 ± 60* 629 ± 299*
9.06 ± 4.62 8.95 ± 2.54
7.02 ± 3.21 * 4.58 ± 1.07**
355 ± 91 * 429 ± 123*
113 ± 57* 108 ± 70*
1011 ± 210* 1294 ± 314*
314 ± 118* 311 ± 206*
11.65 ± 2.30 15.81 ± 4.09
3.19 ± 0.85** 3.97 ± 2.26**
387 ± 54**
85 ± 38*
1302 ± 278*
217± 139*
11.44 ± 4.22
3.80 ± 2.13**
+
# Values are expressed as the mean ± SD.
+ No renal parenchyma.
* Significantly different from sham controls (p <0.05). ** Significantly different from all controls (p <0.003).
weeks was equal to controls who had undergone a nephrectomy (table 1). The post-obstructed (left) kidneys in the experimental groups demonstrated significantly less renal mass than the sham operated controls (p <0.005). Total renal mass in the post-obstructed animals was similar to sham operated controls but significantly greater than either nephrectomy controls or unrelieved obstruction controls (p <0.005). RNA content, DNA content and their ratio (RNA:DNA) were measured to determine if renal growth occurred by hypertrophy or hyperplasia. An increase in RNA content and in the RNA:DNA ratio is consistent with cellular hypertrophy. On the other hand, an increase in DNA content and a decrease in the RNA:DNA ratio are consistent with cellular hyperplasia. The controls undergoing left nephrectomies had significant increases in RNA content and the RNA:DNA ratio (p <0.04). The DNA content was not significantly different from sham operated controls. This is indicative of hypertrophy. The rats undergoing unrelieved left ureteral obstruction showed increases in RNA content at 3 and 6 months, but these increases were not significantly different from sham operated controls. However, the RNA:DNA ratio at 3 months was significantly greater than sham operated controls (p <0.04). This was not true at 6 months. Again, this is consistent with hypertrophic growth. The contralateral kidneys in the experimental groups (obstruction with relief) all showed a different type of response. RNA:DNA ratios were significantly lower (p <0.04) than in sham operated controls, controls undergoing left nephrectomy and controls with left unrelieved ureteral obstruction. As table 2 illustrates, this is due to significant increases in the DNA content, implicating cellular hyperplasia as the main contributor to renal growth. The post-obstructed kidneys in all of the experimental groups at 3 and 6 months demonstrated significant decreases in the RNA:DNA ratios (p <0.007) and significant increases in the DNA content (p <0.01), a reflection of hyperplasia in these kidneys. lnulin clearances measured at 3 and 6 months demonstrated significant decreases (p <0.05) in the post-obstructed kidney at 1, 2 and 3 weeks' obstruction (table 3). The decrease in renal function was directly related to the duration of obstruction. In contrast, the contralateral kidneys showed an increase in func-
TABLE 4. Inulin clearance (GFR) expressed in µl./min./animal for individual kidneys measured at 3 and 6 months for controls and after relief of obstruction for experimental animals.
crnulin (GFR)
Control Sham operated 3 months (no. = 8) 6 months (no. = 8) Left nephrectomy 3 months (no. = 7) 6 months (no. = 7) Left ureteral obstruction 3 months (no. = 7) 6 months (no. = 6) Experimental 1 Wk. obstruction/relieved 3 months (no. = 6) 6 months (no. = 7) 2 Wk. obstruction/relieved 3 months (no. = 7) 6 months (no. = 9) 3 Wk. obstruction/relieved 3 month (no. = 6)
Right
Left
1286 1659
1286 1659
1525 2275 1592 2391 1477 1963
769 1166
2011 2159
629 497
2038
438
tion (inulin and PAH clearances) in all groups. The inulin clearances were significantly increased for rats studied at 6 months (p <0.05). As is depicted in table 3, these increases in inulin clearance were greater, although not quite reaching statistical significance, than the increases associated with left nephrectomies or left unrelieved ureteral obstructions. Effective renal plasma flow (ERPF), as measured by PAH clearance, revealed changes that were very similar to the inulin clearances. The ERPF was significantly decreased (p <0.05) in all of the post-obstructed kidneys in the experimental animals. Again, this was more pronounced with longer periods of obstruction and was true at 3 and 6 months. The contralateral kidneys in the experimental rats demonstrated significant increases in ERPF (p <0.05) for 2 and 3-weeks' obstructed animals at 3 months and 2-weeks' obstructed animals at 6 months. The 1-week obstructed animals did not show significant increases. However, the increase in ERPF in the 2 and 3weeks' obstructed animals was greater than that observed in
1019 the left left unrelieved uretera.l obstruction statis-groups at 3 and 6 rflonths not tical
trnls and unrelieved unilateral obstruction controls achieved cent of a normal GFR. This increase is less only 55 to 60 than most ~~«~"~~ reports. The reason for this discrepancy r,.~,.per 100 grams of body weight. When P.vrm,,:c,Prl per animal, they are 60 to 70 per cent of control These increases are also appreciated by all the experimental groups when calculated in this manner. Thereour data has been presented in a standardized form (per 100 grams body weight). The increased function in the hyperplastic kidneys of the obstructed/relieved animals was greater than that occurring in kidneys which were stimulated to grow hypertrophy (nephrectomy controls and umelieved obstructed controls). This increase in function, although not achieving statistical significance in all experimental animals, is a consistent trend. There is wide variability normally encountered in measuring GFR in rats, suggesting that this trend is significant. The experimental animals who demonstrated an increase in renal mass by hyperplasia had greater renal function than the control animals who had an increase in renal mass by hypertrophy. This implies that the damaged post-obstructed kidney stimulates this hyperplastic growth. A deduction could be made that the post-obstructed kidney produces a substance which is responsible for the contralateral as well as the ipsilateral hyperplastic response, although the data cannot substantiate the humoral nature of the stimulus. Total renal mass is significantly greater in animals in whom obstruction was relieved than in animals where a nephrectomy was performed or who had unrelieved unilateral ureteral obstruction. 1\/i,"""'"'"""'' animals with relieved obstruction had both contralateral and ipsilateral growth by hyperplasia. Renal function as measured by GFR and ERPF was greater for the same dry weight increase if that increase occurred by hyperplasia rather than by hypertrophy (see contralateral kidneys, control vs. experimental, tables 1 and 3). These experiments demonstrate that an obstructed kidney, which will eventually contribute at least 20 per cent to the total renal function, results in better renal function for the whole organism if it is repaired rather than removed. Thus, 1 aspect of the work hypothesis which suggests that a kidney has a greater return in function if it is made to work harder removing its damaged contralateral counterpart is not substantiated by our data. It appears that the post-obstructed, damaged kidney, even though it may add little to overall function, is a major stimulus to improved function in the contralateral kidney. Renal function, as determined GFR, ERPF and osmolar clearance in the post··OtJstruictE:d returned maximally 3 months and not •=·n~r.n~ significantly up to 6 months. that the reduced GFR and ERPF and the im"""-"'··"""-"·J concentrate are permanent in the do not or deteriorate be~ 0
structed/relieved animals were not "'"'"'·'~-,, the controls (table when of the 1, 2 or 3 week respective OSmOlar clearance WaS M•;lHHL,ct!lClY DISCUSSION
Compensatory renal non initiated by many stimuli, including uuuaoc,,m nephrectomy and unilateral ureteral obstruction. 9 · 10 The growth has been shown to be predominantly due to hypertrophy, although some hyperplasia may also occur. 11 Contralateral renal growth after relief of unilateral ureteral obstruction has not been well studied. These experiments demonstrate that relief of varying periods of unilateral ureteral obstruction results in contralateral renal growth by hyperplasia. This response is different from left nephrectomy or left unrelieved obstruction. The absolute increase in renal mass in the contralateral kidney after 2 weeks of obstruction was equal to that after nephrectomy or unrelieved ureteral obstruction when studied at 3 and 6 months. However, the cellular response was r1,1,t1>rPY1t being consistently secondary to The hoTPn<>ran PYT1Pr1,mp also response, -H•w -~"' was associated vvith a ~'F•'"-"vUH decrease in renal mass. It is difficult to assess the actual mechanism of this that a cellular infiltrate in the may play a role in this hyperplastic response rather than the kidney itself. Histologic sections of the post-obstructed kidneys reveal lymphocytic infiltrates which, for the most part, were minimal in extent; however, in rare occasions more extensive infiltrates were observed. Although these infiltrates could alter the RNA and DNA contents and result in a RNA:DNA ratio, the small number of cells in the infiltrates in the of sections makes this unlikely. Since were not seen in the contralateral content observed in these renal units was not due to cytes inadvertently measured with renal cells. It is however, that these infiltrates in the ""'"'··"''"·· may have interacted with the release a substance whid1 could have patterns of the ipsilateral and c.v.""" ,w,wc«u The anir.oals studied at 6 months demonstrated differences 1n The reason for this variation is not clear. It is well known that
there was no variation in the sham controls. In animals at 3 months. The post-obstructed 1 week of obstruction, demonstrated c,1~;111uca11" renal mass with a marked decrease in renal function. the contralateral kidney demonstrated an increase in renal function. The amount of increase, as illustrated in tables 3 and 4, is directly related to the length of obstruction. At 3 months, the post-obstructed kidney function decreases as the period of obstruction increases. The contralateral kidneys have a corresponding increase. This is also evident for the 6-month rats for 1 and 2 weeks' obstruction. Of particular interest is the fact that total renal function is remarkably similar in the 3 obstructed/relieved groups, with total GRF's (inulin clearances) being maintained between 80 and 85 per cent of normal. In contrast, the nephrectomy con-
0 ,,,.,
REFERENCES
l.
B.: Current problems in compensatory renal growth. Bull.
Acad. Med., 50: 1147, 1974. 2. Vaughan, E. D. and Gillenwater, J. Y.: Recovery following complete chronic unilateral ureters! occlusion: functional, radiographic and pathologic alteration. J. Urol., 106: 27, 1971. 3. Van Vroonhoven, T. J., Soler-Montesinos, L. and Malt, R. A.: Humoral regulation of renal mass. Surgery, 72: 300, 1972. 4. Castle, W. N. and McDougal, W. S.: Compensatory renal growth by hyperplasia after relief of chronic unilateral ureteral obstruction. Surg. Forum, 34: 687, 1983. 5. Munro, H. N. and Fleck, A.: The determination of nucleic acids. Methods of Biochemical Analysis, 14: 113, 1966. 6. Burton, K.: A study of the conditions and mechanism of diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J., 62: 315, 1956. 7. Fuhr, J., Kacymarczyk, J. and Kruttgen, C. D.: Eine Einfache Colorimetrische Methode Zur Inulin Betimmung Fur Nieren-
1020
CASTLE AND MCDOUGAL
clearance-Unter-sucaunger Bei Stoffwechselaengenung Und Diubetikern. Klin. Wochenschr., 33: 729, 1955. 8. Bauer, J. D., Ackermann, P. G. and Toro, G.: Brays Clinical Laboratory Methods. St. Louis, C.V. Mosby, 1968. 9. Paulson, D. F. and Fraley, E. E.: Compensatory renal growth after unilateral ureteral obstruction. Kidney Int., 4: 22, 1973.
10. Northrup, R. E. and Malvin, R. L.: Cellular hypertrophy and renal function during compensatory growth. Am. J. Physiol., 231: 1191, 1976. 11. Dicker, S. E. and Shirley, D. G.: Compensatory hypertrophy of the contralateral kidney after unilateral ureteral ligation. J. Physiol., 220: 199, 1972.
THE JOURNAL OF UROLOGY
Copyright© 1984 by The Willia:11s & Wilkins Co.
Printed in U.S.A.
CONTRALATERAL RENAL HYPERPLASIA AND INCREASED RENAL FUNCTION AFTER RELIEF OF CHRONIC UNILATERAL URETERAL OBSTRUCTION WILLIAM N. CASTLE
W. SCOTT MCDOUGAL*
AND
From the Division of Urology, Department of Surgery, Dartmouth Medical School, Hanover, New Hampshire
ABSTRACT
Following relief of 1, 2 or 3 weeks of unilateral ureteral obstruction, contralateral compensatory renal growth and increased renal function were measured 3 and 6 months later. Compensatory growth occurred predominantly by hyperplasia, demonstrated by a significant increase in DNA content and a decrease in the RNA:DNA ratio (p <0.04). This is in contrast to compensatory growth following nephrectomy or unrelieved unilateral ureteral obstruction, which occurred primarily by hypertrophy with no significant change in DNA content but a significant increase in RNA content and the RNA:DNA ratio (p <0.04). Contralateral renal function in animals with relieved unilateral ureteral obstruction was greater than in controls with 2 normal kidneys (p <0.05). The contralateral increase in renal function was greater than that in animals subjected to ipsilateral nephrectomy or unrelieved ureteral obstruction, but this did not reach statistical significance. Thus, when growth occurred by hyperplasia, there was a trend to greater increases in renal function than when growth occurred by hypertrophy. Contralateral compensatory renal hyperplasia and increased renal function occurred in conjunction with a decrease in renal mass and function of the ipsilateral post-obstructed kidney. These experiments suggest that the post-obstructed, poorly functioning kidney stimulates contralateral hyperplastic growth and increased renal function. This hyperplastic response is different from the hypertrophic response following nephrectomy or unrelieved unilateral ureteral obstruction, implicating the post-obstructed kidney as the stimulus of the hyperplastic response. Compensatory renal growth and functional adaptation following chronic unrelieved unilateral ureteral obstruction has been shown to be similar to that following nephrectomy. 1 Growth occurs primarily by cellular hypertrophy with a subsequent increase in renal function. With relief of unilateral ureteral obstruction, the return of function in the post-obstructed kidney is inversely related to the length of time the kidney has been obstructed. 2 However, the compensatory growth response and its relationship to function after relief of obstruction have not been defined. There is evidence to suggest that humoral factors influence renal growth as stimulators or inhibitors of the compensatory response after unilateral nephrectomy or unrelieved ureteral obstruction. 3 These factors have not been identified nor has their site of origin been determined. The following experiments demonstrate that compensatory renal growth following relief of chronic unilateral ureteral obstruction occurs primarily by cellular hyperplasia. 4 This compensatory growth is associated with an increase in renal function. Moreover, the data suggest that the post-obstructed, damaged kidney is the stimulus for this unique response. MATERIALS AND METHODS
One hundred Long Evans male rats weighing between 125 and 150 grams were divided into 6 groups: sham controls, unrelieved unilateral ureteral obstruction, unilateral nephrectomy and unilateral ureteral obstruction relieved by microsurgical ureterovesical reimplantation at 1, 2 and 3 week intervals. All animals were anesthetized with pentobarbital, 50 mg./kg. body weight. Unilateral ureteral obstructions were created through Accepted for publication June 4, 1984. * Requests for reprints: Dept. of Urology, Vanderbilt University Medical Center North, Nashville, TN 37232. Read at annual meeting of the American Urological Association, New Orleans, Louisiana, May 10, 1984.
a midline incision by placing an occlusive ligature about the left ureter at the ureterovesical junction. Left nephrectomies were performed through a flank incision. Sham controls underwent a midline incision without manipulation of ureters. Rats undergoing relief of unilateral ureteral obstruction were divided into 3 subgroups: 1, 2 and 3-weeks' obstruction. At the end of the period of obstruction, the animals were anesthetized with pentobarbital (50 mg./kg. body weight), and the obstructed ureter was reimplanted into the bladder using 8-0 collagen (Ethicon) and an operating microscope. Control animals were not subjected to sham operations at this time. After all operations, the rats were placed in individual cages and allowed free access to rat chow and deionized water. All animals were allowed to recover for either 3 or 6 months, except for the 3-weeks' obstructed group which was studied only at 3 months. Following the 3 or 6 month recovery period, the rats were again anesthetized with pentobarbital, 50 mg./kg. body weight. They were placed on a thermoregulated table, and the carotid artery, jugular vein and both ureters (or 1 ureter and the bladder) were cannulated using PE-10 or PE-50 tubing. All reimplanted ureters were unobstructed; their diameters were greatly reduced as compared to their size at the time of reim plantation by approximately 80 to 90 per cent. An infusion of 10 per cent Inutest (Laevosan-Gesselschaft) and 0.5 per cent para-amino hippurate (PAH) (Merck, Sharp, and Dome) in lactated Ringer's solution was infused through the jugular vein at a rate of 10 ,ul./min./100 gm. body weight. Each rat also received a bolus of lactated Ringer's (equal to 2 per cent of his body weight) at the beginning of the infusion to replace evaporative losses during the procedure. The rats were allowed to equilibrate for 45 minutes with this infusion, followed by a 60minute collection of urine obtained under oil. At the midpoint and endpoint of the urine collection, a blood sample was obtained via the carotid artery. The experiment was terminated
1016
1017
CONTRALATERAL REt-;AL f-IYPERPLASIA
both and determining wet in pretins. One half of each kidney was prepared immediately determination by the modified Schmidt-Thannhauser for procedure,5 and for DNA determination by the diphenylamine reaction. 6 The other half of each kidney was placed in a preweighed tin, minced and dried at 37C for 3 days to determine
renal dry •Neight. Blood and urine were analyzed for inulin by the An throne Method, 7 P AH by the Bratton-Marshall reaction8 and osmolality by freezing point depression. Statistical analyses were performed using Student's t test. A level of p < 0.05 was assumed significant. RESULTS
L Renal dry weight (mg./100 gm. body weight) for individual kidneys measured at 3 and 6 months for control and experimental animals#
TABLE
Percent Change in Dry Weight Compared to Sham Operated Rats
Renal Dry Weight
Right Control Sham operated 3 months (no.= 10) 6 months (no. = 13) Left nephrectomy 3 months (no. = 8) 6 months (no. = 9) Left ureteral obstruction 3 months (no. = 7) 6 months (no. = 9) Experimental l Week obstruction/ relieved 3 months (no. = 8) 6 months (no. = 8) 2 Weeks obstruction/ relieved 3 months (no. = 11) 6 months (no. = 10) 3 Weeks obstruction/ relieved 3 months (no. = 6)
7.0 ± 0.6 6.7 ± 0.5
Left
Right
Left
6.6 ± 0.7 6.7 ± 0.6
10.3 ± 0.5* 9.8 ± 0.8*
+47 +46
9.3 ± 0.9* 9.1 ± 0.5*
+ +
+33 +36
8.2 ± 0.7** 7.2 ± 0.9
5.2 ± 1.1* 4.3 ± 1.1 *
+17 + 7
-36
10.2 ± 1.6* 9.2 ± 1.8*
3.9 ± 1.3* 3.0 ± 1.9
+46 +37
-41 -55
9.1 ± 0.7'''
2.7 ± 0.8*
+30
-59
-21
# Values are expressed as the mean ± SD. + No renal parenchyma. * Significantly different from sham controls (p <0.05). ** Significantly different from all controis (p <0.005).
TABLE 2.
Body weights at the time of clearance measurements were not significantly different for groups studied at 3 months. Sham operated controls were 493 ± 69 gm. (mean ± SD), 502 ± 49 gm. for left nephrectomy controls, and 504 ± 35 gm. for left unrelieved ureteral obstruction controls. Body weights for the experimental animals at 3 months were 502 ± 51 gm. for 1week obstructions, 495 ± 69 gm. for 2-weeks' obstructions and 514 ± 43 gm. for 3-weeks' obstructions. Body weights for the rats studied at 6 months demonstrated some variation. Sham operated controls weighed 537 ± 45 gm., left nephrectomy controls 637 ± 29 gm. and unrelieved left ureteral obstruction controls 603 ± 62 gm. The latter 2 groups are significantly different from sham operated controls (p <0.007). Body weights for the experimental animals at 6 months were 589 ± 57 gm. for I-week obstructions, which is significantly different from sham operated controls (p <0.02), and 525 ± 69 gm. for 2weeks' obstructions, which is not significantly different from the sham operated controls. Renal dry weights (table 1) demonstrated significant changes in renal mass. All groups, except the 1-week obstructed rats studied at 6 months, were significantly different from sham operated controls (p <0.005). The left nephrectomy and left unrelieved ureteral obstruction controls had significantly greater renal dry weights at 3 and 6 months (p <0.005). This is consistent with compensatory renal growth. There was no discernible renal parenchyma (histologically) in the left unrelieved ureteral obstructed kidney at 3 or 6 months. The increases in renal mass in the contralateral kidney of the nephrectomy and unrelieved ureteral obstruction controls were significantly greater (p <0.005) than those seen in the contralateral kidney in the 1-week obstructed rats at 3 and 6 months. However, there was no significant difference with longer periods of obstruction, at 2 and 3 weeks. The contralateral renal growth in animals whose obstruction was relieved at 2 and 3
RNA content, DNA content (mg./gm. renal weight), and RNA:DNA ratio for individual kidneys measured at 3 and 6 months for controls and after relief of obstruction for experimental animals# DNA Content
RNA Content Right Control Sham operated 3 months (no. 6 months (no.= Left nephrectomy 3 months (no. = 8) 6 months (no. = 9) Left ureteral obstruction 3 months (no. = 7) 6 months (no. = 9) Experimental 1 Week obstruction/ relieved 3 months (no. = 8) 6 months (no. = 8) 2 Weeks obstruction/ relieved 3 months (no. = 11) 6 months (no. = 10) 3 Weeks obstruction/ relieved 3 months (no. = 6)
40.40 ± 1.86 42.81 ± 2.90
Left
40.45 ± 3.37 42.86 ± 2.50
48.52 ± 7.12* 46.65 ± 6.46*
Right
22.75 ± 2.57 23.40 ± 3.29
RNA:DNA Left
22.19 ± 1.74 22.70 ± 2.91
21.86 ± 5.54 21.67 ± 3.45
42.82 ± 4.25 43.34 ± 2.89
+ +
20.76 ± 4.50 23.59 ± 1.66
Right
1. 794 ± 0.195
l.856 ± 0.240
Left
1.829 ± 0.180 1.916 ± 0.261
2.397 ± 0.803* 2.194 ± 0.455*
+ +
2.135 ± 0.363* 1.843 ± 0.148
+ +
42.1 7 ± 3.42** 41.03 ± 3.80**
42.69 ± 6.21 41.46 ± 4.16
27.02 ± 3.55**'' 25.58 ± 4.97**
28.05 ± 3.82* 31.81 ± 8.77*
1.653 ± 0.209** 1.682 ± 0.477
1.556 ± 0.241 * 1.401 ± 0.4 72*
34.65 ± 6.29*** 41.38 ± 3.45**
33.60 ± 6.48* 42.15 ± 4.35
26.34 ± 7.42*** 28.69 ± 5.89***
30.84 ± 5.93* 35.14 ± 7.15*
1.374 ± 0.300** 1.505 ± 0.346**
1.129 ± 0.307* 1.256 ± 0.322*
32.46 ± 4.28***
30.45 ± 1.62*
27.00 ± 3.73***
28. 75 ± 4.12*
1.327 ± 0.252**
1.051 ± 0.125*
# Values are expressed as the mean ± SD. + No renal parenchyma. * Significantly different from sham controls (p <0.05). ** Significantly different from control nephrectomies (p <0.03). *** Significantly different from all controls (p <0.005).
1018
CASTLE AND MCDOUGAL
TABLE 3.
Inulin, para-amino hippurate and osmolar clearances (µl./min./100 gm. body weight) for individual kidneys measured at 3 and 6 months for controls and after relief of obstruction for experimental animals# crnulin (GFR) Right
cosm
cPAH (ERPF)
(Osmolar Clearance)
Left
Right
Left
Right
285 ± 67 325 ± 97
856 ± 211 1075 ± 314
856 ± 211 1075 ± 314
10.36 ± 2.61 10.27 ± 2.63
Left
Control Sham operated 3 months (no. = 8) 6 months (no.= 8) Left nephrectomy 3 months (no. = 7) 6 months (no. = 7) Left ureteral obstruction 3 months (no. = 7) 6 months (no. = 6) Experimental 1 Week obstruction/ relieved 3 months (no. = 6) 6 months (no. = 7) 2 Weeks obstruction/ relieved 3 months (no. = 7) 6 months (no. = 9) 3 Weeks obstruction/ relieved 3 months (no. = 6)
285 ± 67 325 ± 97 303 ± 32* 359 ± 83
9.74 ± 2.01 10.27 ± 2.63
7.06 ± 2.47 14.67 ± 3.93
1164 ± 306* 1126 ± 377
311 ± 117 402 ± 92*
+ +
968 ± 255 1195 ± 322
+ +
9.59 ± 5.04 15.29 ± 4.88
+
309 ± 80 345 ± 145
159 ± 43* 204 ± 75*
1052 ± 349 960 ± 434
492 ± 60* 629 ± 299*
9.06 ± 4.62 8.95 ± 2.54
7.02 ± 3.21 * 4.58 ± 1.07**
355 ± 91 * 429 ± 123*
113 ± 57* 108 ± 70*
1011 ± 210* 1294 ± 314*
314 ± 118* 311 ± 206*
11.65 ± 2.30 15.81 ± 4.09
3.19 ± 0.85** 3.97 ± 2.26**
387 ± 54**
85 ± 38*
1302 ± 278*
217± 139*
11.44 ± 4.22
3.80 ± 2.13**
+
# Values are expressed as the mean ± SD.
+ No renal parenchyma.
* Significantly different from sham controls (p <0.05). ** Significantly different from all controls (p <0.003).
weeks was equal to controls who had undergone a nephrectomy (table 1). The post-obstructed (left) kidneys in the experimental groups demonstrated significantly less renal mass than the sham operated controls (p <0.005). Total renal mass in the post-obstructed animals was similar to sham operated controls but significantly greater than either nephrectomy controls or unrelieved obstruction controls (p <0.005). RNA content, DNA content and their ratio (RNA:DNA) were measured to determine if renal growth occurred by hypertrophy or hyperplasia. An increase in RNA content and in the RNA:DNA ratio is consistent with cellular hypertrophy. On the other hand, an increase in DNA content and a decrease in the RNA:DNA ratio are consistent with cellular hyperplasia. The controls undergoing left nephrectomies had significant increases in RNA content and the RNA:DNA ratio (p <0.04). The DNA content was not significantly different from sham operated controls. This is indicative of hypertrophy. The rats undergoing unrelieved left ureteral obstruction showed increases in RNA content at 3 and 6 months, but these increases were not significantly different from sham operated controls. However, the RNA:DNA ratio at 3 months was significantly greater than sham operated controls (p <0.04). This was not true at 6 months. Again, this is consistent with hypertrophic growth. The contralateral kidneys in the experimental groups (obstruction with relief) all showed a different type of response. RNA:DNA ratios were significantly lower (p <0.04) than in sham operated controls, controls undergoing left nephrectomy and controls with left unrelieved ureteral obstruction. As table 2 illustrates, this is due to significant increases in the DNA content, implicating cellular hyperplasia as the main contributor to renal growth. The post-obstructed kidneys in all of the experimental groups at 3 and 6 months demonstrated significant decreases in the RNA:DNA ratios (p <0.007) and significant increases in the DNA content (p <0.01), a reflection of hyperplasia in these kidneys. lnulin clearances measured at 3 and 6 months demonstrated significant decreases (p <0.05) in the post-obstructed kidney at 1, 2 and 3 weeks' obstruction (table 3). The decrease in renal function was directly related to the duration of obstruction. In contrast, the contralateral kidneys showed an increase in func-
TABLE 4. Inulin clearance (GFR) expressed in µl./min./animal for individual kidneys measured at 3 and 6 months for controls and after relief of obstruction for experimental animals.
crnulin (GFR)
Control Sham operated 3 months (no. = 8) 6 months (no. = 8) Left nephrectomy 3 months (no. = 7) 6 months (no. = 7) Left ureteral obstruction 3 months (no. = 7) 6 months (no. = 6) Experimental 1 Wk. obstruction/relieved 3 months (no. = 6) 6 months (no. = 7) 2 Wk. obstruction/relieved 3 months (no. = 7) 6 months (no. = 9) 3 Wk. obstruction/relieved 3 month (no. = 6)
Right
Left
1286 1659
1286 1659
1525 2275 1592 2391 1477 1963
769 1166
2011 2159
629 497
2038
438
tion (inulin and PAH clearances) in all groups. The inulin clearances were significantly increased for rats studied at 6 months (p <0.05). As is depicted in table 3, these increases in inulin clearance were greater, although not quite reaching statistical significance, than the increases associated with left nephrectomies or left unrelieved ureteral obstructions. Effective renal plasma flow (ERPF), as measured by PAH clearance, revealed changes that were very similar to the inulin clearances. The ERPF was significantly decreased (p <0.05) in all of the post-obstructed kidneys in the experimental animals. Again, this was more pronounced with longer periods of obstruction and was true at 3 and 6 months. The contralateral kidneys in the experimental rats demonstrated significant increases in ERPF (p <0.05) for 2 and 3-weeks' obstructed animals at 3 months and 2-weeks' obstructed animals at 6 months. The 1-week obstructed animals did not show significant increases. However, the increase in ERPF in the 2 and 3weeks' obstructed animals was greater than that observed in
1019 the left left unrelieved uretera.l obstruction statis-groups at 3 and 6 rflonths not tical
trnls and unrelieved unilateral obstruction controls achieved cent of a normal GFR. This increase is less only 55 to 60 than most ~~«~"~~ reports. The reason for this discrepancy r,.~,.per 100 grams of body weight. When P.vrm,,:c,Prl per animal, they are 60 to 70 per cent of control These increases are also appreciated by all the experimental groups when calculated in this manner. Thereour data has been presented in a standardized form (per 100 grams body weight). The increased function in the hyperplastic kidneys of the obstructed/relieved animals was greater than that occurring in kidneys which were stimulated to grow hypertrophy (nephrectomy controls and umelieved obstructed controls). This increase in function, although not achieving statistical significance in all experimental animals, is a consistent trend. There is wide variability normally encountered in measuring GFR in rats, suggesting that this trend is significant. The experimental animals who demonstrated an increase in renal mass by hyperplasia had greater renal function than the control animals who had an increase in renal mass by hypertrophy. This implies that the damaged post-obstructed kidney stimulates this hyperplastic growth. A deduction could be made that the post-obstructed kidney produces a substance which is responsible for the contralateral as well as the ipsilateral hyperplastic response, although the data cannot substantiate the humoral nature of the stimulus. Total renal mass is significantly greater in animals in whom obstruction was relieved than in animals where a nephrectomy was performed or who had unrelieved unilateral ureteral obstruction. 1\/i,"""'"'"""'' animals with relieved obstruction had both contralateral and ipsilateral growth by hyperplasia. Renal function as measured by GFR and ERPF was greater for the same dry weight increase if that increase occurred by hyperplasia rather than by hypertrophy (see contralateral kidneys, control vs. experimental, tables 1 and 3). These experiments demonstrate that an obstructed kidney, which will eventually contribute at least 20 per cent to the total renal function, results in better renal function for the whole organism if it is repaired rather than removed. Thus, 1 aspect of the work hypothesis which suggests that a kidney has a greater return in function if it is made to work harder removing its damaged contralateral counterpart is not substantiated by our data. It appears that the post-obstructed, damaged kidney, even though it may add little to overall function, is a major stimulus to improved function in the contralateral kidney. Renal function, as determined GFR, ERPF and osmolar clearance in the post··OtJstruictE:d returned maximally 3 months and not •=·n~r.n~ significantly up to 6 months. that the reduced GFR and ERPF and the im"""-"'··"""-"·J concentrate are permanent in the do not or deteriorate be~ 0
structed/relieved animals were not "'"'"'·'~-,, the controls (table when of the 1, 2 or 3 week respective OSmOlar clearance WaS M•;lHHL,ct!lClY DISCUSSION
Compensatory renal non initiated by many stimuli, including uuuaoc,,m nephrectomy and unilateral ureteral obstruction. 9 · 10 The growth has been shown to be predominantly due to hypertrophy, although some hyperplasia may also occur. 11 Contralateral renal growth after relief of unilateral ureteral obstruction has not been well studied. These experiments demonstrate that relief of varying periods of unilateral ureteral obstruction results in contralateral renal growth by hyperplasia. This response is different from left nephrectomy or left unrelieved obstruction. The absolute increase in renal mass in the contralateral kidney after 2 weeks of obstruction was equal to that after nephrectomy or unrelieved ureteral obstruction when studied at 3 and 6 months. However, the cellular response was r1,1,t1>rPY1t being consistently secondary to The hoTPn<>ran PYT1Pr1,mp also response, -H•w -~"' was associated vvith a ~'F•'"-"vUH decrease in renal mass. It is difficult to assess the actual mechanism of this that a cellular infiltrate in the may play a role in this hyperplastic response rather than the kidney itself. Histologic sections of the post-obstructed kidneys reveal lymphocytic infiltrates which, for the most part, were minimal in extent; however, in rare occasions more extensive infiltrates were observed. Although these infiltrates could alter the RNA and DNA contents and result in a RNA:DNA ratio, the small number of cells in the infiltrates in the of sections makes this unlikely. Since were not seen in the contralateral content observed in these renal units was not due to cytes inadvertently measured with renal cells. It is however, that these infiltrates in the ""'"'··"''"·· may have interacted with the release a substance whid1 could have patterns of the ipsilateral and c.v.""" ,w,wc«u The anir.oals studied at 6 months demonstrated differences 1n The reason for this variation is not clear. It is well known that
there was no variation in the sham controls. In animals at 3 months. The post-obstructed 1 week of obstruction, demonstrated c,1~;111uca11" renal mass with a marked decrease in renal function. the contralateral kidney demonstrated an increase in renal function. The amount of increase, as illustrated in tables 3 and 4, is directly related to the length of obstruction. At 3 months, the post-obstructed kidney function decreases as the period of obstruction increases. The contralateral kidneys have a corresponding increase. This is also evident for the 6-month rats for 1 and 2 weeks' obstruction. Of particular interest is the fact that total renal function is remarkably similar in the 3 obstructed/relieved groups, with total GRF's (inulin clearances) being maintained between 80 and 85 per cent of normal. In contrast, the nephrectomy con-
0 ,,,.,
REFERENCES
l.
B.: Current problems in compensatory renal growth. Bull.
Acad. Med., 50: 1147, 1974. 2. Vaughan, E. D. and Gillenwater, J. Y.: Recovery following complete chronic unilateral ureters! occlusion: functional, radiographic and pathologic alteration. J. Urol., 106: 27, 1971. 3. Van Vroonhoven, T. J., Soler-Montesinos, L. and Malt, R. A.: Humoral regulation of renal mass. Surgery, 72: 300, 1972. 4. Castle, W. N. and McDougal, W. S.: Compensatory renal growth by hyperplasia after relief of chronic unilateral ureteral obstruction. Surg. Forum, 34: 687, 1983. 5. Munro, H. N. and Fleck, A.: The determination of nucleic acids. Methods of Biochemical Analysis, 14: 113, 1966. 6. Burton, K.: A study of the conditions and mechanism of diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J., 62: 315, 1956. 7. Fuhr, J., Kacymarczyk, J. and Kruttgen, C. D.: Eine Einfache Colorimetrische Methode Zur Inulin Betimmung Fur Nieren-
1020
CASTLE AND MCDOUGAL
clearance-Unter-sucaunger Bei Stoffwechselaengenung Und Diubetikern. Klin. Wochenschr., 33: 729, 1955. 8. Bauer, J. D., Ackermann, P. G. and Toro, G.: Brays Clinical Laboratory Methods. St. Louis, C.V. Mosby, 1968. 9. Paulson, D. F. and Fraley, E. E.: Compensatory renal growth after unilateral ureteral obstruction. Kidney Int., 4: 22, 1973.
10. Northrup, R. E. and Malvin, R. L.: Cellular hypertrophy and renal function during compensatory growth. Am. J. Physiol., 231: 1191, 1976. 11. Dicker, S. E. and Shirley, D. G.: Compensatory hypertrophy of the contralateral kidney after unilateral ureteral ligation. J. Physiol., 220: 199, 1972.