Reversible Inner Medullary Vascular Obstruction in Acute Experimental Hydronephrosis Melvin M. Schwartz, MD, Manjeri A. Venkatachalam, MB, BS, and Ramzi S. Cotran, MD
The pathogenesis of experimental unilateral hydronephrosis was studied in the rat, with emphasis on the role of medullary vascular obstruction. The medullary circulation was evaluated after increasing periods of ureteral obstruction (up to 48 hours) using in vivo perfusion of silicone rubber or colloidal carbon. Evidence of inner medullary hypoperfusion was seen after 1 hour, and by 6 hours, the entire papilla was ischemic. Papillarv hypoperfusion was still present at 24 and 48 hours, but significant recirculation occurred in the presence of continued obstruction. Release of ureteral obstruction caused rapid reversal of the perfusion defect. Histologic studies of the renal parenchyma adjacent to the pelvis showed increasing vascular congestion, focal interstitial edema, and extravasation of erythrocytes; these changes were also diminished after release of obstruction. Endothelial swelling or thrombosis in inner medullary blood vessels was not observed. Inner medullary tubules showed degenerative changes in kidneys that had been obstructed for 6 hours or more; at later periods (18 hours or more) focal necrosis was seen, but it never involved the entire papilla. Thus, reversible collapse of inner medullary blood vessels occurs in acute unilateral hydronephrosis and may provide the basis for the development of ischemic tubular damage. The findings suggest that the vascular obstruction may be due to increased intrapelvic pressure, rather than endothelial swelling or thrombosis. The vascular-tubular defect may be relevant to the altered medullary physiology observed in this condition. (Am J Pathol 86:425-436, 1977)
A VARIETY OF DEFECTS in cortical perfusion and function have been described during and after release of acute ureteral obstruction.'5 The first morphologic changes, however, are manifest in the medullarv parenchyma and peripelvic tissues,6 and it has been shown that concentrating ability is impaired even after short periods of hvdronephrosis." 5'7 It has been suggested that early medullarv damage following ureteral obstruction may be due to impaired medullary circulation,6'8 but physiologic studies have reported conflicting results.7'F12 In this study, injection techniques were used to evaluate the patency of the medullary vasculature during and after release of acute unilateral obstruction; light and electron microscopy were used to document the extent of tissue necrosis and to correlate it with the occurrence of perFrom the Department of Pathology, Peter Bent Brigham Hospital and Harvard Medical School. Boston, Massachusetts. Supported bv Grants HL-08251 and ANM-16749 from the National Institutes of Health. Accepted for publication September 20, 1976. Address reprint requests to Dr. Ramzi S. Cotran, Department of Pathology, Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, MA 02115. 425
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fusion defects. The results provide a demonstration of acute reversible reduction of inner medullary perfusion in the early stages of unilateral ureteral obstruction.
Materiqls and Methods Animals
Seventy-two Sprague Dawley rats of either sex were used. The animals were given standard laboratory chow and water ad libitum and weighed 100 to 300 g prior to the experiments. Surgical Procedure
The animals were anesthetized with sodium pentobarbital (30 to 40 mg/kg intraperitoneally), supplemented with ether. The left ureter was isolated through a midline incision and was obstructed with a silk ligature approximately midway between the renal hilus and the urinary bladder. Reversible ureteral obstruction was produced by isolating the ureter at the same point and ligating it against a piece of polyethylene tubing with umbilical tape. After a timed interval, the animal was reanesthetized, and the tape was carefully removed from the ureter. When immediate release of obstruction was required, the ureter was separated from the surrounding adipose tissue and divided above the ligature. Care was taken not to disturb the kidney during the procedure. Testing of Vascular Patency
Vascular patency was tested by the infusion of opaque media. In one group of rats, colloidal carbon (Biological Ink, Pelikan Co., Hannover, West Germany, Batch No. cll/1431a) was injected intravenously in doses of 2.5 ml/100 g body weight, following the technique of Strock and Majno,18 and the animals were sacrificed immediately therafter. The renal vessels were clamped and kidneys fixed by immersion overnight in neutral buffered formalin. Slices of fixed tissue were either cleared in graded glycerol solution or embedded in paraffin by routine methods for histologic examination. Areas of hypoperfusion were seen grossly as white areas; on histologic examination, unperfused vessels contained no carbon. A second group was perfused with a silicone rubber compound (Microfil, Canton Biomedical Products, Boulder, Colo.) via an aortic cannula connected to a constant pressure reservoir."' Blood flow to the organ was carefully maintained until perfusion with silicone rubber at 150 mm Hg had begun. Perfusion was continued until the injection medium flowed freely from the incised vena cava. Following dehydration in alcohol and clearing in methyl salicylate, handcut slices were observed with a dissecting microscope for completeness of perfusion. Quantitation of Histopathology Histologic changes were seen and quantitated as follows: 1 + tissue damage was the mildest lesion and consisted of peripelvic and periforniceal tubular necrosis with interstitial hemorrhage and edema; 2+ damage showed, in addition, focal areas of tubular necrosis in the inner medulla; 3+ damage was the most severe and included areas of neutrophilic exudation in foci of inner medullary necrosis.
Quantitation of Perfusion Defects Perfusion defects in the renal medulla were quantitated as follows: 1+ defects were confined to the superficial peripelvic and periforniceal tissues; 2+ defects included patchy
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disruption of filling within the inner medulla; and 3+ defects were the most severe, with total lack of inner medullary perfusion.
_ Desp Kidneys from two groups of animals were examined (Table 1). In Group I, continuous ureteral obstruction was maintained for periods of 15 minutes to 48 hours. In Group II, obstruction was maintained for period of 15 minutes to 48 hours, and kidneys were examined either immediately (Group IIA) or 24 hours (Group IIB) after release of the ureteral ligature. Numbers of animals examined in each group and time intervals are shown in Table 1. Elron For electron microscopy, the kidneys were fixed after release of ureteral obstruction bv intraarterial perfusion 14 with 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4," for 20 minutes after a preliminary rinse of the vascular tree with lactated Ringer's solution for 1 minute. In other experiments, renal tissue was also fixed by immersion for 3 hours at room temperature in the same fixative. Tissue was selected from grossly abnormal areas as well as from successively deeper medullarv levels in both the obstructed kidney and its unobstructed control. Afte postfixation in 1% aqueous osmium tetroxide, the tissue was rapidly dehydrated in alcohol and embedded in Epon 812. One-micron-thick sections were stained with 1% toluidine blue in 1% aqueous borax while thin sections were stained with uranyl acetate and lead citrate and examined in a Philips 200 electron microscope.
Table 1-Summary of Observations on Obstructed Kidney Duration of obstuction 15min 1hr 2hrs 4hrs 6hrs 12hrs 18hrs 24hrs 48hrs Histopathology (Groups I 0 0 0 0 + ++ +++ +++ +++ and 11) (14) (5) (2) (4) (2) (4) (4) (4) (4) Perfusion defects with
continued obstructiont (Group I)
Perfusion defects after release of obstructon Immediately after
0
+
+
++
+++
++
++
++
++
(1)
(1)
(2)
(3)
(9)
(4)
(3)
(10)
(5)
0 (6)
reease (Group IIA) Twenty-four hours after rease 0 (Group IIB)
0
0
0
0
(1)
(1)
(1)
(1)
(1)
0
(4)
+, Focal peripevic necrosis; ++, focal papillary necrosis; +++, focal papillary necrosis with neutrophils. t +, Peripelvc and fomiceal perfusion defect + +, papilla partially perfused; + ++, papilla
severely hypoperfused. Figures in parentheses are number of observations at each interval. Control unobstructed kidneys did not exhibit tissue damage or perfusion defects (0 score).
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Results Gross and Microscopic Pathology
The gross and microscopic features after total obstruction were similar to those reported previously 5,6 and will be only briefly described. At 15 minutes there was mild tubular dilatation and forniceal vascular congestion without hydronephrosis or tubular degenerative changes. After 1 hour, in addition to tubular dilatation, early hydronephrosis and mild cortical vascular congestion were present. Between 2 and 6 hours, hydronephrosis and cortical congestion became prominent. After 6 hours, areas of red blood cell extravasation and interstitial edema were seen in the fornices, in the vicinity of congested veins and capillaries (Figure 1). In addition, the first signs of tubular degeneration were evident. This was a focal finding, and consisted of individual cell swelling, vacuolization, and necrosis in the papillary tissue adjacent to the fornices. Examination of kidneys 24 hours following release of ureteral ligation revealed no further progression in the expression of tubular injury beyond that observed during or immediately following release of obstruction. At 12 hours, necrotic tubules and microinfarcts were observed in the papilla. By 18 hours, tubular dilatation and vascular congestion were maximal. Papillary tubular necrosis and microinfarcts (Figure 2A) were frequently accompanied by mild neutrophilic exudates (Figure 2B). Pelvic dilatation was further increased after 24 and 48 hours, but total necrosis or atrophy of the papilla was never observed. Observations by electron microscopy largely confirmed the focal nature of the epithelial cell degeneration seen by light microscopy and offered no new information on the pathogenesis of the lesion. The inner medullary vasculature showed no evidence of thrombosis or endothelial swelling. Injection Studies
The findings with silicone rubber and colloidal carbon were similar and are illustrated in Figures 3-6. Continued Obstruction
As compared with the extent of vascular filling in unobstructed right kidneys, the inner medullae of obstructed left kidneys (Group I, Table 1) developed a progressive perfusion defect. At 1 and 2 hours it was confined to the superficial papillary tissue adjacent to the fornices. At 4 hours there was patchy hypoperfusion throughout the inner medulla. By 6 hours, it was no longer possible to fill any but occasional vasa recta or capillaries in the inner medulla (Figures 3A, 4, and 5A) as compared with the control
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right kidneys (Figures 3B and 5B). In histologic sections and cleared tissue slices, all glomeruli, including juxtamedullary glomeruli, appeared perfused. No gross filling defects were observed in the postglomerular capillary plexus of the cortex or outer medulla, but the vasa recta exhibited a complete cut-off near the inner-outer medullary junction. Adequacy of perfusion (in the functional sense) could not be judged by the images obtained; these provided information only with respect to the presence of complete vascular obstruction. When the obstruction period was extended beyond 6 hours and the completeness of perfusion studied at 12, 18, 24, and 48 hours (Table 1), there was partial perfusion of the papilla in the presence of continued obstruction (Figure 6A). Although the extent of reflow varied from animal to animal, it never approached the completeness of filling seen in control right kidneys (Figure 6B), and by inference, a significant medullary perfusion defect persisted.
AfterRPelse of brtion Reversibility of the perfusion defect was studied by infusing carbon and silicone rubber immediately or 24 hours following release of the ureteral ligature. When kidneys obstructed for periods up to 6 hours were studied 24 hours after release of ligature (Group II, Table 1), no perfusion defects were observed in the inner medulla. The time actually required for return of flow was exceedingly short, for if hydronephrosis established for 6 or 24 hours was relieved immediately before perfusion, patency of the inner medullary vasculature was reestablished (Figures 3C and 6C). In separate experiments, if both ureters were ligated and only one released, then the inner medulla of the released kidney perfused and that of the obstructed kidney did not. As before, the adequacy of cortical and outer medullary filling could not be evaluated quantitatively during this postobstructive period; the images obtained suggested that there were no large filling defects similar to those seen in obstructed inner medullae. The inherent limitations of the technique of study by injection of specimens precluded such quantitation of the extent of flow (see below). Discussion
The results indicate that acute ureteral obstruction causes a rapid reversible decrease in inner medullary vascular perfusion and, further, that with continued obstruction (up to 48 hours) there is partial restoration of vascular patency. The degree of vascular filling in the cortex and outer medulla during continued obstruction, as well as in the postobstructive period, could not
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be quantitated and appeared to be influenced partially by section thickness. The injection techniques used by us in the present protocol only indicated patency of the vessels studied; increase in vascular tone could not be judged due to a) shrinkage of vascular casts by the clearing process used (alcohol and methyl salicylate) and b) masking of arteries and arterioles by filled postglomerular plexus capillaries. Study of the renal vasculature in established hydronephrosis has revealed the occurrence of vascular abnormalities such as stretching and kinking of intrarenal arteries and venous compression.6'17 The sequential parenchymal changes that lead to established hydronephrosis have been attributed to these vascular lesions as well as to increased tissue pressure.61"6"7 However, the early vascular events in hydronephrosis have received less attention. Disturbances in renal cortical function, attributed to preglomerular vasoconstriction, occur after release of unilateral ureteral obstruction lasting 24 hours.2'8 The occurrence of such a vascular defect was suggested by correlated morphologic studies using incomplete silicone rubber injection in vivo.3 Reduction of renal cortical blood flow, probably due to preglomerular vasoconstriction, has been shown to occur also during continued ureteral obstruction of 24 hours' duration without release.' Measurement of regional blood flow to the medulla in hydronephrosis has been complicated by the relative inaccessibility of the medulla and the anatomy of its blood supply. The medulla receives such a small fraction of the total renal blood flow that even marked fluctuation in its perfusion may be masked by the preponderant cortical component.'8 Thus, most physiologic studies have depended upon indirect and often unreliable methods of blood flow measurement, with some studies supporting and others denying reduction of renal medullary blood flow in acute hydronephrosis.79'12 The most recent study in.rats, using the intravenous 125I albumin infusion technique,10 has shown a progressive decline in inner medullary plasma flow to 22% of normal within 18 hours of unilateral obstruction and a rapid return toward normal (69% to 78% of controls) 2 hours following relief of obstruction. Our qualitative morphologic observations with regard to inner medullary perfusion by colloidal carbon and silicone rubber are in close agreement with the functional data obtained by Solez et al.'0 Thus, inner medullary filling defects were apparent by 2 hours, became maximal by 6 hours following obstruction, and were reversed by release of ureteral pressure. Our in vivo findings are also consistent with the in vitro experiments of Muirhead et al.8 In this study, excised dog kidneys were perfused with India ink suspension during elevation of intrapelvic hydrostatic pressure through a water column;
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decreased filling of inner medullary blood vessels was observed to result. The initial cessation of blood flow to the inner medulla in the acutely obstructed kidney may have caused ischemic tissue damage and cell swelling in the endothelium of nonperfused vessels. Ischemia-mediated endothelial cell swelling (and consequent narrowing of vascular lumen) has been shown to cause the so-called no-reflow phenomenon following release of 1 and 2 hours' total renal artery oc-clusion in rats.1' Although focal microscopic areas of tubular degeneration and necrosis did develop in inner medullary tissue within 48 hours of persistent obstruction, the vast majority of inner medullary blood vessels could be demonstrated to be patent and free of significant endothelial swelling following release of ureteral obstruction. These vascular patency studies, in conjunction with the functional data obtained by Solez et al.,'o suggest that a significant noreflow phenomenon, at least of the proportions seen in the postischemic kidney, does not develop in the inner medulla of the postobstructed rat kidney. By inference, then, the cessation of inner medullary perfusion in acute ureteral obstruction may be attributed predominantly to compression of the relatively thin walled medullary capillaries by raised intrapelvic pressure. The above considerations may also be relevant to our observation that in the face of continued obstruction (during the 6- to 48-hour period) there was partial restoration of inner medullary vascular patency to colloidal carbon and silicone rubber. Thus, in previous studies on unilateral ureteral obstruction in rats, intrapelvic pressure rose to 54 mm Hg by 6 hours, but fell to 32 mm Hg by 18 hours in the face of persistent obstruction."0 Similar observations were made in the dog kidney.21 Regardless of the mechanisms responsible for this decline in intrapelvic pressure, such as decreased glomerular filtration or escape of pelvic urine via lymphatics or veins, the consequent partial restoration of vascular patency may help explain why focal necrosis, rather than total infarction, developed in the affected inner medulla during the first 48 hours. The subsequent events that lead to continued medullary and cortical destruction and atrophy in chronic hydronephrosis have been discussed previously.5'"'1"7 Briefly, these include pressure atrophy, continued renal ischemia due to vasomotor changes, arterial obstruction due to stretching and kinking, as well as venous obstruction. Our studies shed light only on the inner medullary hemodynamic events immediately following obstruction and may be relevant to the ensuing occurrence of focal inner medullary necrosis. These degenerative changes in the inner medulla may explain, at least in part, the loss of urinary concentrating ability that is observed during or after acute hydronephrosis.1'.7
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References 1. Vaughan ED Jr, Sorenson EJ, Gillenwater JY: Effects of acute and chronic ureteral obstruction on renal hemodynamics and function. Surg Forum 19:536-538, 1968 2. Jaenike JR: The renal response to ureteral obstruction: A model for the study of factors which influence glomerular filtration pressure. J Lab Clin Med 76:373-382, 1970
3. Harris RH, Yarger WE: Renal function after release of unilateral ureteral obstruction in rats. Am J Physiol 227:806-815, 1974 4. McDougal WS, Wright FS: Defect in proximal and distal sodium transport in post obstructive diuresis. Kidney Int 2:304-317, 1972 5. Nagle RB, Bulger RE, Cutler RE, Jervis HR, Benditt EP: Unilateral obstructive nephropathy in the rabbit. I. Early morphologic, physiologic and histochemical changes. Lab Invest 28:456467, 1973 6. Sheehan HL, Davis JC: Experimental hydronephrosis. Arch Pathol 68:185-225, 1959 7. Honda N, Aizawa C, Morikawa A, Yoshitoshi Y: Effect of elevated ureteral pressure on renal medullary osmolal concentration in hydropenic rabbits. Am J Physiol 221:698-703, 1971 8. Muirhead EE, Vanatta J, Grollman A: Papillary necrosis of the kidney: A clinical and experimental, correlation. JAMA 142:627-631, 1950 9. Harsing L, Szanto G, Bartha J: Renal circulation during stop flow in the dog. Am J Physiol 213:935-938, 1967 10. Solez K, Vernon N, Finer PM, Miller M, Heptinstall RH: Medullary plasma flow and ureteral pressure in unilateral and bilateral ureteral obstruction. Kidney Int 6:99a, 1974 (Abstr) 11. Selkurt EE: Effect of ureteral blockade on renal blood flow and urinary concentrating ability. Am J Physiol 205:286-292, 1963 12. Suki WN, Guthrie AG, Martinez-Maldonado M, Eknoyan G: Effects of ureteral pressure elevation on renal hemodynamics and urine concentration. Am J Physiol 220:3843, 1971 13. Strock PE, Majno G: Vascular responses to experimental tourniquet ischemia. Surg Gynecol Obstet 129:309-318, 1969 14. Griffith LD, Bulger RE, Trump BF: The ultrastructure of the functioning kidney. Lab Invest 16:220-246, 1967 15. Karnovsky MJ: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy, J Cell Biol 27:137a-138a, 1965 16. Hinman F: Hydronephrosis. I. The structural changes. Surgery 17:816-835, 1945 17. Rao NR, Heptinstall RH: Experimental hydronephrosis: A microangiographic study. Invest Urol 6:183-204, 1968 18. Thurau K, Levine DZ: The renal circulation. The Kidney: Morphology, Biochemistry, Physiology, Vol III. Edited by C Rouiller, AF Muller. New York, Academic Press, Inc., 1971, pp 1-70 19. Flores J, DiBona DR, Beck CH, Leaf A: The role of cell swelling in ischemic renal damage and the protective effect of hypertonic solute. J Clin Invest 51:118-126, 1972 20. Summers WK, Jamison RL: The no reflow phenomenon in renal ischemia. Lab Invest 25:635-643, 1971 21. Kiil F: The Function of Ureter and Renal Pelvis. Philadelphia, W. B. Saunders, 1957
Acknowledgments Technical assistance was provided by D. Sandstrom and secretarial assistance by P. M. Higgins. Dr. Schwartz's present address is Department of Pathology, Rush-Presbyterian-St. Luke's Medical Center, 1953 West Congress Parkway, Chicago, IL 60612.
F.r 1-Rat renal papilla after 6 hours' ureterai ligation. Dilated blood vessels and iterstitial hemorrhage are present in th peripesvic tissue at the pehMc fornix (F). (Epon embedded, toluldine blue stain, x 750) Fg. 2A and B-Rat renal papilla after 18 hours of ureteral liaton. A-Focal area of ischemc necrosis (N) in the papilla and adanwt to the pevic fomix (F) (H&E, X 250). Engorged blood vessels (upper left) and polymorphonucear leukocytes (arrow) are present at the margin of the infarct (H&E, x 750).
2A
3B
3A
-A
The pathogenesis of experimental unilateral hydronephrosis was studied in the rat, with emphasis on the role of medullary vascular obstruction. The medullary circulation was evaluated after increasing periods of ureteral obstruction (up to 48 hours) using in vivo perfusion of silicone rubber or colloidal carbon. Evidence of inner medullary hypoperfusion was seen after 1 hour, and by 6 hours, the entire papilla was ischemic. Papillarv hypoperfusion was still present at 24 and 48 hours, but significant recirculation occurred in the presence of continued obstruction. Release of ureteral obstruction caused rapid reversal of the perfusion defect. Histologic studies of the renal parenchyma adjacent to the pelvis showed increasing vascular congestion, focal interstitial edema, and extravasation of erythrocytes; these changes were also diminished after release of obstruction. Endothelial swelling or thrombosis in inner medullary blood vessels was not observed. Inner medullary tubules showed degenerative changes in kidneys that had been obstructed for 6 hours or more; at later periods (18 hours or more) focal necrosis was seen, but it never involved the entire papilla. Thus, reversible collapse of inner medullary blood vessels occurs in acute unilateral hydronephrosis and may provide the basis for the development of ischemic tubular damage. The findings suggest that the vascular obstruction may be due to increased intrapelvic pressure, rather than endothelial swelling or thrombosis. The vascular-tubular defect may be relevant to the altered medullary physiology observed in this condition. (Am J Pathol 86:425-436, 1977)
A VARIETY OF DEFECTS in cortical perfusion and function have been described during and after release of acute ureteral obstruction.'5 The first morphologic changes, however, are manifest in the medullarv parenchyma and peripelvic tissues,6 and it has been shown that concentrating ability is impaired even after short periods of hvdronephrosis." 5'7 It has been suggested that early medullarv damage following ureteral obstruction may be due to impaired medullary circulation,6'8 but physiologic studies have reported conflicting results.7'F12 In this study, injection techniques were used to evaluate the patency of the medullary vasculature during and after release of acute unilateral obstruction; light and electron microscopy were used to document the extent of tissue necrosis and to correlate it with the occurrence of perFrom the Department of Pathology, Peter Bent Brigham Hospital and Harvard Medical School. Boston, Massachusetts. Supported bv Grants HL-08251 and ANM-16749 from the National Institutes of Health. Accepted for publication September 20, 1976. Address reprint requests to Dr. Ramzi S. Cotran, Department of Pathology, Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, MA 02115. 425
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fusion defects. The results provide a demonstration of acute reversible reduction of inner medullary perfusion in the early stages of unilateral ureteral obstruction.
Materiqls and Methods Animals
Seventy-two Sprague Dawley rats of either sex were used. The animals were given standard laboratory chow and water ad libitum and weighed 100 to 300 g prior to the experiments. Surgical Procedure
The animals were anesthetized with sodium pentobarbital (30 to 40 mg/kg intraperitoneally), supplemented with ether. The left ureter was isolated through a midline incision and was obstructed with a silk ligature approximately midway between the renal hilus and the urinary bladder. Reversible ureteral obstruction was produced by isolating the ureter at the same point and ligating it against a piece of polyethylene tubing with umbilical tape. After a timed interval, the animal was reanesthetized, and the tape was carefully removed from the ureter. When immediate release of obstruction was required, the ureter was separated from the surrounding adipose tissue and divided above the ligature. Care was taken not to disturb the kidney during the procedure. Testing of Vascular Patency
Vascular patency was tested by the infusion of opaque media. In one group of rats, colloidal carbon (Biological Ink, Pelikan Co., Hannover, West Germany, Batch No. cll/1431a) was injected intravenously in doses of 2.5 ml/100 g body weight, following the technique of Strock and Majno,18 and the animals were sacrificed immediately therafter. The renal vessels were clamped and kidneys fixed by immersion overnight in neutral buffered formalin. Slices of fixed tissue were either cleared in graded glycerol solution or embedded in paraffin by routine methods for histologic examination. Areas of hypoperfusion were seen grossly as white areas; on histologic examination, unperfused vessels contained no carbon. A second group was perfused with a silicone rubber compound (Microfil, Canton Biomedical Products, Boulder, Colo.) via an aortic cannula connected to a constant pressure reservoir."' Blood flow to the organ was carefully maintained until perfusion with silicone rubber at 150 mm Hg had begun. Perfusion was continued until the injection medium flowed freely from the incised vena cava. Following dehydration in alcohol and clearing in methyl salicylate, handcut slices were observed with a dissecting microscope for completeness of perfusion. Quantitation of Histopathology Histologic changes were seen and quantitated as follows: 1 + tissue damage was the mildest lesion and consisted of peripelvic and periforniceal tubular necrosis with interstitial hemorrhage and edema; 2+ damage showed, in addition, focal areas of tubular necrosis in the inner medulla; 3+ damage was the most severe and included areas of neutrophilic exudation in foci of inner medullary necrosis.
Quantitation of Perfusion Defects Perfusion defects in the renal medulla were quantitated as follows: 1+ defects were confined to the superficial peripelvic and periforniceal tissues; 2+ defects included patchy
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disruption of filling within the inner medulla; and 3+ defects were the most severe, with total lack of inner medullary perfusion.
_ Desp Kidneys from two groups of animals were examined (Table 1). In Group I, continuous ureteral obstruction was maintained for periods of 15 minutes to 48 hours. In Group II, obstruction was maintained for period of 15 minutes to 48 hours, and kidneys were examined either immediately (Group IIA) or 24 hours (Group IIB) after release of the ureteral ligature. Numbers of animals examined in each group and time intervals are shown in Table 1. Elron For electron microscopy, the kidneys were fixed after release of ureteral obstruction bv intraarterial perfusion 14 with 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4," for 20 minutes after a preliminary rinse of the vascular tree with lactated Ringer's solution for 1 minute. In other experiments, renal tissue was also fixed by immersion for 3 hours at room temperature in the same fixative. Tissue was selected from grossly abnormal areas as well as from successively deeper medullarv levels in both the obstructed kidney and its unobstructed control. Afte postfixation in 1% aqueous osmium tetroxide, the tissue was rapidly dehydrated in alcohol and embedded in Epon 812. One-micron-thick sections were stained with 1% toluidine blue in 1% aqueous borax while thin sections were stained with uranyl acetate and lead citrate and examined in a Philips 200 electron microscope.
Table 1-Summary of Observations on Obstructed Kidney Duration of obstuction 15min 1hr 2hrs 4hrs 6hrs 12hrs 18hrs 24hrs 48hrs Histopathology (Groups I 0 0 0 0 + ++ +++ +++ +++ and 11) (14) (5) (2) (4) (2) (4) (4) (4) (4) Perfusion defects with
continued obstructiont (Group I)
Perfusion defects after release of obstructon Immediately after
0
+
+
++
+++
++
++
++
++
(1)
(1)
(2)
(3)
(9)
(4)
(3)
(10)
(5)
0 (6)
reease (Group IIA) Twenty-four hours after rease 0 (Group IIB)
0
0
0
0
(1)
(1)
(1)
(1)
(1)
0
(4)
+, Focal peripevic necrosis; ++, focal papillary necrosis; +++, focal papillary necrosis with neutrophils. t +, Peripelvc and fomiceal perfusion defect + +, papilla partially perfused; + ++, papilla
severely hypoperfused. Figures in parentheses are number of observations at each interval. Control unobstructed kidneys did not exhibit tissue damage or perfusion defects (0 score).
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Results Gross and Microscopic Pathology
The gross and microscopic features after total obstruction were similar to those reported previously 5,6 and will be only briefly described. At 15 minutes there was mild tubular dilatation and forniceal vascular congestion without hydronephrosis or tubular degenerative changes. After 1 hour, in addition to tubular dilatation, early hydronephrosis and mild cortical vascular congestion were present. Between 2 and 6 hours, hydronephrosis and cortical congestion became prominent. After 6 hours, areas of red blood cell extravasation and interstitial edema were seen in the fornices, in the vicinity of congested veins and capillaries (Figure 1). In addition, the first signs of tubular degeneration were evident. This was a focal finding, and consisted of individual cell swelling, vacuolization, and necrosis in the papillary tissue adjacent to the fornices. Examination of kidneys 24 hours following release of ureteral ligation revealed no further progression in the expression of tubular injury beyond that observed during or immediately following release of obstruction. At 12 hours, necrotic tubules and microinfarcts were observed in the papilla. By 18 hours, tubular dilatation and vascular congestion were maximal. Papillary tubular necrosis and microinfarcts (Figure 2A) were frequently accompanied by mild neutrophilic exudates (Figure 2B). Pelvic dilatation was further increased after 24 and 48 hours, but total necrosis or atrophy of the papilla was never observed. Observations by electron microscopy largely confirmed the focal nature of the epithelial cell degeneration seen by light microscopy and offered no new information on the pathogenesis of the lesion. The inner medullary vasculature showed no evidence of thrombosis or endothelial swelling. Injection Studies
The findings with silicone rubber and colloidal carbon were similar and are illustrated in Figures 3-6. Continued Obstruction
As compared with the extent of vascular filling in unobstructed right kidneys, the inner medullae of obstructed left kidneys (Group I, Table 1) developed a progressive perfusion defect. At 1 and 2 hours it was confined to the superficial papillary tissue adjacent to the fornices. At 4 hours there was patchy hypoperfusion throughout the inner medulla. By 6 hours, it was no longer possible to fill any but occasional vasa recta or capillaries in the inner medulla (Figures 3A, 4, and 5A) as compared with the control
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right kidneys (Figures 3B and 5B). In histologic sections and cleared tissue slices, all glomeruli, including juxtamedullary glomeruli, appeared perfused. No gross filling defects were observed in the postglomerular capillary plexus of the cortex or outer medulla, but the vasa recta exhibited a complete cut-off near the inner-outer medullary junction. Adequacy of perfusion (in the functional sense) could not be judged by the images obtained; these provided information only with respect to the presence of complete vascular obstruction. When the obstruction period was extended beyond 6 hours and the completeness of perfusion studied at 12, 18, 24, and 48 hours (Table 1), there was partial perfusion of the papilla in the presence of continued obstruction (Figure 6A). Although the extent of reflow varied from animal to animal, it never approached the completeness of filling seen in control right kidneys (Figure 6B), and by inference, a significant medullary perfusion defect persisted.
AfterRPelse of brtion Reversibility of the perfusion defect was studied by infusing carbon and silicone rubber immediately or 24 hours following release of the ureteral ligature. When kidneys obstructed for periods up to 6 hours were studied 24 hours after release of ligature (Group II, Table 1), no perfusion defects were observed in the inner medulla. The time actually required for return of flow was exceedingly short, for if hydronephrosis established for 6 or 24 hours was relieved immediately before perfusion, patency of the inner medullary vasculature was reestablished (Figures 3C and 6C). In separate experiments, if both ureters were ligated and only one released, then the inner medulla of the released kidney perfused and that of the obstructed kidney did not. As before, the adequacy of cortical and outer medullary filling could not be evaluated quantitatively during this postobstructive period; the images obtained suggested that there were no large filling defects similar to those seen in obstructed inner medullae. The inherent limitations of the technique of study by injection of specimens precluded such quantitation of the extent of flow (see below). Discussion
The results indicate that acute ureteral obstruction causes a rapid reversible decrease in inner medullary vascular perfusion and, further, that with continued obstruction (up to 48 hours) there is partial restoration of vascular patency. The degree of vascular filling in the cortex and outer medulla during continued obstruction, as well as in the postobstructive period, could not
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be quantitated and appeared to be influenced partially by section thickness. The injection techniques used by us in the present protocol only indicated patency of the vessels studied; increase in vascular tone could not be judged due to a) shrinkage of vascular casts by the clearing process used (alcohol and methyl salicylate) and b) masking of arteries and arterioles by filled postglomerular plexus capillaries. Study of the renal vasculature in established hydronephrosis has revealed the occurrence of vascular abnormalities such as stretching and kinking of intrarenal arteries and venous compression.6'17 The sequential parenchymal changes that lead to established hydronephrosis have been attributed to these vascular lesions as well as to increased tissue pressure.61"6"7 However, the early vascular events in hydronephrosis have received less attention. Disturbances in renal cortical function, attributed to preglomerular vasoconstriction, occur after release of unilateral ureteral obstruction lasting 24 hours.2'8 The occurrence of such a vascular defect was suggested by correlated morphologic studies using incomplete silicone rubber injection in vivo.3 Reduction of renal cortical blood flow, probably due to preglomerular vasoconstriction, has been shown to occur also during continued ureteral obstruction of 24 hours' duration without release.' Measurement of regional blood flow to the medulla in hydronephrosis has been complicated by the relative inaccessibility of the medulla and the anatomy of its blood supply. The medulla receives such a small fraction of the total renal blood flow that even marked fluctuation in its perfusion may be masked by the preponderant cortical component.'8 Thus, most physiologic studies have depended upon indirect and often unreliable methods of blood flow measurement, with some studies supporting and others denying reduction of renal medullary blood flow in acute hydronephrosis.79'12 The most recent study in.rats, using the intravenous 125I albumin infusion technique,10 has shown a progressive decline in inner medullary plasma flow to 22% of normal within 18 hours of unilateral obstruction and a rapid return toward normal (69% to 78% of controls) 2 hours following relief of obstruction. Our qualitative morphologic observations with regard to inner medullary perfusion by colloidal carbon and silicone rubber are in close agreement with the functional data obtained by Solez et al.'0 Thus, inner medullary filling defects were apparent by 2 hours, became maximal by 6 hours following obstruction, and were reversed by release of ureteral pressure. Our in vivo findings are also consistent with the in vitro experiments of Muirhead et al.8 In this study, excised dog kidneys were perfused with India ink suspension during elevation of intrapelvic hydrostatic pressure through a water column;
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decreased filling of inner medullary blood vessels was observed to result. The initial cessation of blood flow to the inner medulla in the acutely obstructed kidney may have caused ischemic tissue damage and cell swelling in the endothelium of nonperfused vessels. Ischemia-mediated endothelial cell swelling (and consequent narrowing of vascular lumen) has been shown to cause the so-called no-reflow phenomenon following release of 1 and 2 hours' total renal artery oc-clusion in rats.1' Although focal microscopic areas of tubular degeneration and necrosis did develop in inner medullary tissue within 48 hours of persistent obstruction, the vast majority of inner medullary blood vessels could be demonstrated to be patent and free of significant endothelial swelling following release of ureteral obstruction. These vascular patency studies, in conjunction with the functional data obtained by Solez et al.,'o suggest that a significant noreflow phenomenon, at least of the proportions seen in the postischemic kidney, does not develop in the inner medulla of the postobstructed rat kidney. By inference, then, the cessation of inner medullary perfusion in acute ureteral obstruction may be attributed predominantly to compression of the relatively thin walled medullary capillaries by raised intrapelvic pressure. The above considerations may also be relevant to our observation that in the face of continued obstruction (during the 6- to 48-hour period) there was partial restoration of inner medullary vascular patency to colloidal carbon and silicone rubber. Thus, in previous studies on unilateral ureteral obstruction in rats, intrapelvic pressure rose to 54 mm Hg by 6 hours, but fell to 32 mm Hg by 18 hours in the face of persistent obstruction."0 Similar observations were made in the dog kidney.21 Regardless of the mechanisms responsible for this decline in intrapelvic pressure, such as decreased glomerular filtration or escape of pelvic urine via lymphatics or veins, the consequent partial restoration of vascular patency may help explain why focal necrosis, rather than total infarction, developed in the affected inner medulla during the first 48 hours. The subsequent events that lead to continued medullary and cortical destruction and atrophy in chronic hydronephrosis have been discussed previously.5'"'1"7 Briefly, these include pressure atrophy, continued renal ischemia due to vasomotor changes, arterial obstruction due to stretching and kinking, as well as venous obstruction. Our studies shed light only on the inner medullary hemodynamic events immediately following obstruction and may be relevant to the ensuing occurrence of focal inner medullary necrosis. These degenerative changes in the inner medulla may explain, at least in part, the loss of urinary concentrating ability that is observed during or after acute hydronephrosis.1'.7
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References 1. Vaughan ED Jr, Sorenson EJ, Gillenwater JY: Effects of acute and chronic ureteral obstruction on renal hemodynamics and function. Surg Forum 19:536-538, 1968 2. Jaenike JR: The renal response to ureteral obstruction: A model for the study of factors which influence glomerular filtration pressure. J Lab Clin Med 76:373-382, 1970
3. Harris RH, Yarger WE: Renal function after release of unilateral ureteral obstruction in rats. Am J Physiol 227:806-815, 1974 4. McDougal WS, Wright FS: Defect in proximal and distal sodium transport in post obstructive diuresis. Kidney Int 2:304-317, 1972 5. Nagle RB, Bulger RE, Cutler RE, Jervis HR, Benditt EP: Unilateral obstructive nephropathy in the rabbit. I. Early morphologic, physiologic and histochemical changes. Lab Invest 28:456467, 1973 6. Sheehan HL, Davis JC: Experimental hydronephrosis. Arch Pathol 68:185-225, 1959 7. Honda N, Aizawa C, Morikawa A, Yoshitoshi Y: Effect of elevated ureteral pressure on renal medullary osmolal concentration in hydropenic rabbits. Am J Physiol 221:698-703, 1971 8. Muirhead EE, Vanatta J, Grollman A: Papillary necrosis of the kidney: A clinical and experimental, correlation. JAMA 142:627-631, 1950 9. Harsing L, Szanto G, Bartha J: Renal circulation during stop flow in the dog. Am J Physiol 213:935-938, 1967 10. Solez K, Vernon N, Finer PM, Miller M, Heptinstall RH: Medullary plasma flow and ureteral pressure in unilateral and bilateral ureteral obstruction. Kidney Int 6:99a, 1974 (Abstr) 11. Selkurt EE: Effect of ureteral blockade on renal blood flow and urinary concentrating ability. Am J Physiol 205:286-292, 1963 12. Suki WN, Guthrie AG, Martinez-Maldonado M, Eknoyan G: Effects of ureteral pressure elevation on renal hemodynamics and urine concentration. Am J Physiol 220:3843, 1971 13. Strock PE, Majno G: Vascular responses to experimental tourniquet ischemia. Surg Gynecol Obstet 129:309-318, 1969 14. Griffith LD, Bulger RE, Trump BF: The ultrastructure of the functioning kidney. Lab Invest 16:220-246, 1967 15. Karnovsky MJ: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy, J Cell Biol 27:137a-138a, 1965 16. Hinman F: Hydronephrosis. I. The structural changes. Surgery 17:816-835, 1945 17. Rao NR, Heptinstall RH: Experimental hydronephrosis: A microangiographic study. Invest Urol 6:183-204, 1968 18. Thurau K, Levine DZ: The renal circulation. The Kidney: Morphology, Biochemistry, Physiology, Vol III. Edited by C Rouiller, AF Muller. New York, Academic Press, Inc., 1971, pp 1-70 19. Flores J, DiBona DR, Beck CH, Leaf A: The role of cell swelling in ischemic renal damage and the protective effect of hypertonic solute. J Clin Invest 51:118-126, 1972 20. Summers WK, Jamison RL: The no reflow phenomenon in renal ischemia. Lab Invest 25:635-643, 1971 21. Kiil F: The Function of Ureter and Renal Pelvis. Philadelphia, W. B. Saunders, 1957
Acknowledgments Technical assistance was provided by D. Sandstrom and secretarial assistance by P. M. Higgins. Dr. Schwartz's present address is Department of Pathology, Rush-Presbyterian-St. Luke's Medical Center, 1953 West Congress Parkway, Chicago, IL 60612.
F.r 1-Rat renal papilla after 6 hours' ureterai ligation. Dilated blood vessels and iterstitial hemorrhage are present in th peripesvic tissue at the pehMc fornix (F). (Epon embedded, toluldine blue stain, x 750) Fg. 2A and B-Rat renal papilla after 18 hours of ureteral liaton. A-Focal area of ischemc necrosis (N) in the papilla and adanwt to the pevic fomix (F) (H&E, X 250). Engorged blood vessels (upper left) and polymorphonucear leukocytes (arrow) are present at the margin of the infarct (H&E, x 750).
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