Immunization Against Retrograde Pyelonephritis I

Immunization Against Retrograde Pyelonephritis I. Production of an Experimental Model of Severe Ascending Escherichia coli Pyelonephritis Without Bacteremia in Rats

S. J. D. Brooks, MD, J. M. Lyons, MS and A. 1. Braude, MD

Retrograde pyelonephritis was produced in rats by introducing into the bladder a small refluxing inoculum of Escherichia coli that would enter the renal pelvis but not the blood stream. At the same time the left ureter was partially obstructed for 18 hours. This model differs from previous attempts to produce E coli pyelonephritis with large (0.6 ml) volumes infused into the bladder, because such large volumes cause bacteremia and hematogeneous pyelonephritis. Since retrograde E coli pyelonephritis in patients is not accompanied by positive blood cultures, the model described in this report is believed to accurately mimic human pyelonephritis and to allow a realistic approach to the study of immunity against retrograde infection in the urinary tract (Am J Pathol 74:345-358, 1974).

IN VIEW OF THE FAILURE of antibacterial drugs to prevent pyelonephritis, another approach must be taken to prevent this disease. A promising alternative to drugs is immunization, but before this can be undertaken it is necessarv to develop an experimental model that reproduces human pyelonephritis. Despite many attempts to reproduce human pyelonephritis in animals, fewv, if any, have succeeded in creating an experimental model of retrograde E coli pyelonephritis. Since E coli is by far the main cause of human pyelonephritis and the ascending route the most common, a truly representative model of the disease in man should result in the spread of E coli to the kidney from the bladder 'with the development of suppurative renal lesions. Infection of the kidney has been produced after inoculating E coli into the bladder of mice undergoing water diuresis, but gross pyelonephritic lesions have not been described wvith consistency and no attempt has been made to exclude hematogenous infection.',2 Pyelonephritic lesions occur in rats given larger bladder infusions of E coli via the urethra,3 4 but

From the Departments of Medicine and Pathology, University of California, San Diego, School of Medicine, San Diego, Calif. Supported by Research Grant 5R01 HE-12967-04 and Training Grant 5T01-A1- 00391-05 from the US Public Health Service. Accepted for publication October 17, 1973. Address reprint requests to Dr. Abraham Braude, Department of Medicine, Universitv of California, San Diego, University Hospital, 225 West Dickinson St, San Diego, CA 92103. 345 346 BROOKS ET AL American Journal of Pathology Fierer5 demonstrated that such large volumes opened pvelovenous communications and caused bacteremia leading to hematogenous infection of the kidney. To produce retrograde pvelonephritis in rats without bacteremia, we introduced a small refluxing inoculum of E coli into the bladder that would enter the pelvis but not the blood stream. At the same time we produced partial temporary* ureteral obstruction. We believe that this is the first experimental model that accuratelv mirrors human E coli pvelonephritis and allows a realistic approach to the studv of immunitv against retrograde infection in the urinary tract.

Materials and Methods Male Sprague-Dawlev rats (Holtzmann Laboratories) weighing 180 to 240 g were deprived of water for 18 hours overnight, because previous studies 5 have shown that reflux is unpredictable in animals allowed to drink freely. With the rats under ether anesthesia, the left ureter was exposed through a midline incision of the abdominal wall. At midureter level, a rounded No. 10 half circle needle, threaded with size 0 black braided silk was passed through the flank and then successively posterior, medial and anterior to the left ureter. The left flank was again pierced, this time from the inside, so that the needle came through onlv a few millimeters from the original site of penetration. The ligature was left loosely in place while the bladder was visualized and infused with 0.4 cc of a 106 dilution of the overnight culture in trypticase soy broth (TSB) (Difco). The bladder was alwavs completelv emptv immediatehl before the infusion because of water deprivation, and because shaving the operative sites and ether anesthesia encouraged micturition. Two smooth strains of E coli of human origin were used. The strain of E coli 0:111 B4 was nonmotile, serum sensitive and nonhemolytic, whereas E coli 06 (Riffle) was motile, serum resistant and hemolytic. Both are K antigen positive. The strain of E cdo 0:11 B4 was selected for study because it is one of the best characterized strains of E coli from the standpoint of immunochemical properties and pathogenicity for the urinarv tract. E coli 06 (Riffle), obtained from 'Marvin Turck, was isolated from a human urinary tract infection. The organisms were stored on a trypticase soy agar slant (TSA) (Difco), and a loopful was inoculated into TSB and incubated overnight at 37 C. A 106 dilution was made the following day in TSB. The number of viable bacteria in the inoculum was determined by serial dilutions and plate counts. A volume of 0.4 ml of the bacterial suspension was infused via a tuberculin syringe, with a 27-gauge needle, into the bladder over a period of 30 seconds. Once the infusion was completed, the two arms of the ureteral ligature were tied loosely. Before the ligature was knotted, the ureter was inspected, so that any, excessive kInldng or contact with the abdominal wall could be corrected. The abdominal wall was closed in a single layer with braided silk sutures. Approximately 0.3 ml of blood was taken from the tail, 3 minutes after infusion, by the "farmer's wife" method.6 One-tenth ml in TSA was used to make pour plates and the rest of the blood used for antibody studies, carried out bv the hemagglutination technic. Tap water was retumed to the animals postoperatively. Approximately 20 hours later, the animals were again ether-anesthetized, and the knot of the ligature gently freed from the skin. One arm of the ligature was cut Vol. 74, No. 2 RETROGRADE PYELONEPHRITIS 347 February 1974 from the outside, and the ligature gently removed, first by pinching up the skin under the knot and finally by pulling out the ligature. At this time the swollen kidney was seen bulging in the left flank. The animals were killed at various times after the original operation. Immediately before termiinal anesthesia, the penis was clamped to prevent loss of urine and the rat exsanguinated by heart puncture to obtain blood for cultures and agglutination tests. The dead animals were dipped in Zephirang solution, and their abdornens were opened. Observations were made of the ureter at the site of ligature, the presence of hydroureter, and the presence of cvstitis or prostatic abscesses. The kidneys were removed and placed in sterile containers, and the bladder urine aspirated with a tuberculin syringe. The kidneys were observed for enlargement and gross abscesses. The area occupied by the abscess was graded on a scale of 1 to 8, each point on the scale representing one eighth of the total kidney surface. The kidneys were sectioned longitudinally and the presence of hydronephrosis and fresh pus in the pelvis noted. The whole kidney was homogenized unless a portion was used for micro- scopic studies, and a loopful of the homogenate streaked onto 5% Blood Azide (Difco), and eosin-methvlene-blue (EMB) (L)ifco) plates. They were incubated for 48 hours at 37 C and graded by a method previously described.7 This is a variation of the parallel streak method. A loopful of the material is inoculated on to one quadrant of the plate. The second, third and fourth series were each made at right angles to the previous one, so that the original inoculum was progressively diluted. The bacteriologic grade was determined bv noting whether the growth extended into the first, second, third or fourth quadrant. The urines were treated likewise. Throughout the experiment, colonies were typed against specific anti- 0:111 or 06 serum to establish the identity of the infecting organism.8 The serum antibody response was measured by the hemagglutination of red cells, to which E coli lipopolysaccharide was absorbed by a modification of the Neter method.9 The lipopolysaccharides of E coli 06 (Riffle) and E coli 0:111 B4 were extracted by the boivin method.9 Sera from each infected rat was examined for antibody against lipopolysaccharide from the strain of E coli responsible for his infection. For histologic study the kidneys were sectioned longitudinally, preserved in formalin, and subsequently stained with hematoxylin and eosin. Three or four sections were cut from each kidney, except at 36 hours when serial sections were done. A random selection of nonligated kidneys from each group were also examined histologically.

Results Six hours after inoculation of E coli 06 (Riffle), the kidneys were greatly enlarged but with only minimal hydronephrosis and no inflam- matory areas. Grossly visible abscesses were first seen at 20 hours in rats infected with E coli 06 but not until 36 hours in those infected with E coi 0:111. This difference in speed of development and severity of lesions was maintained throughout the 72-hour period of comparison between the two serotypes (Table 1). At 72 hours the pyelonephritic abscesses were more than twice as extensive with E coli 06. These lesions extended from the cortex through the medulla and riddled the inter- stitial areas with suppuration (Figure 1). 348 BROOKS ET AL American Journal of Pathology

Table 1-Comparative Severity of Pyelonephritis Due to E Coli 06 Riffle and E Coli 0:111 Strain of Average EColi Time Incidence % grade* 0:111 20 hours 0/20 0 0 36 hours 10/15 67 1 72 hours 35/42 80 2.5 06 Riffle 20 hours 7/10 70 1 72 hours 7/10 70 5.7 * Each point on the grade scale equals one eight of the total kidney surface occupied by an abscess.

Although gross renal lesions developed faster and became more severe in the rats infected with E coli 06, their pattern of development was the same in both groups. The earliest lesions were abscesses composed of polymorphonuclear leukocytes (PMN) in the anterior, posterior and superior fornices (Figure 2). Those in the superior fornix extended into the outer medulla and upper papilla but never into the lower third of the papilla and its tip (Figure 3). They also spread into the cortex where they produced abscesses protruding beyond the surface (Fig- ure 1). Moderate external and internal hydronephrosis persisted throughout the first 72 hours despite free flow through the ureter into the bladder. The evolution of pyelonephritis was observed in animals infected with E coli 0: 111 for 1 month. Observations were made at 8, 15 and 28 days on 16, 18 and 12 rats, respectively. Gross pyelonephritis at each interval was found in 67 to 87¶ of the animals and invariably on the side of the temporarily kinked ureter. At 8 days the major change was the appearance of lymphocytes and plasma cells in large numbers, although PMINs still predominated. Hvdronephrosis was also still present. E coli 0:111 was isolated from all but one pyelonephritic kidney, but the degree of infection had dropped from a grade of 3.4 to 1.7. At 15 days the lymphocyte had become the predominant cell, with PMINs still present especially in the pus casts. Plasma cells and large mononuclear cells resembling macrophages were also seen. At this time E coli 0:111 could be cultured only from one-third of the pyelonephritic kidneys, and the severity of infection in this group averaged only 1.1. In other words, the change in pattern of cellular reaction to a predominance of lympho- cvtes was associated with sterilization of the infection. At this time the bulging cortical abscesses have given way to shalloxv cortical depressions coincident with the disappearance of acute suppuration. At 28 days the infection had disappeared but left shrunken scarred Vol. 74, No. 2 RETROGRADE PYELONEPHRITIS 349 February 1974 kidneys, with persistent areas of internal hydronephrosis but no external hydronephrosis. The scars were a mixture of fibrosis and chronic inflam- matory cells, especially lymphocytes and plasma cells. They extended from the medulla to the cortex but never to the papillary tip or central papilla. Ureteral and Pelvic Lesions Intravenous pyelograms performed when the ligature was in place and just before sacrifice at 3 days showed free flow of dye through the ureter into the bladder with evidence of hydronephrosis above the ligature (Figure 4). Ureteritis and severe purulent pyelitis occurred in rats infected with E coli 06 but not in those with E coli 0: 111. A reaction to the ligature occurred regularly in the periureteral ad- ventitia but this did not interfere with urine flow.

The Nonligated Side Gross pyelonephritis never developed in the right kidney. Slight PMN infiltration of the fornices was occasionally seen at 20 hours but this disappeared by 36 hours. Despite the absence of gross or micro- scopic lesions of pyelonephritis, kidneys on the nonligated side became infected, but less frequently and with fewer organisms than in the pyelonephritic kidneys (Table 2).

Prostatic Abscesses At 72 hours, grossly visible prostatic abscesses were found in 38% of 42 rats infected with E coli 0:111 and 40! of the 10 infected with E coli

Table 2-The Effect of Loose Ureteral Ligature on Incidence of Bacteria in the Kidneys- The Nonligated Kidneys Gave Positive Cultures in the Complete Absence of Pyelonephritis Loose ligature No ligature Strain of E Coli Time Incidence Grade* Incidence Grade* 0:111 20 hours 20/20 3.7 12/20 2.5 36 hours 12/15 3.75 8/15 1.5 72 hours 37/42 3.40 24/42 1.3 8 days 10/16 1.70 3/16 1.0 15 days 5/18 1.10 2/18 1.0 28 days 0/12 0 0/12 0 0.6 Riffle 6 hours 8/10 3.0 8/10 2.0 20 hours 10/10 3.6 8/10 2.9 72 hours 9/10 3.3 9/10 3.0 Bacteriologic grade determined by parallel streak method, as described in the text. 350 BROOKS ET AL American Journal of Pathology 06. At 15 days, 50% of 18 rats had prostatic abscesses. Histologically these abscesses resembled those previously reported in Proteus prostatitis by Friedlander and Braude.0

Bacteriogic Results Viable bacterial counts showed that the pyrelonephritic lesions were produced by inoculating 50 to 100 organisms of either serotype into the bladder. All rats were subjected to blood culture 3 minutes after bladder inoculation, and bacteremia was never found. After-vards, among 35 animals infected wvith E coli 0:111 and subjected to blood cultures at either 20 or 36 hours, no bacteremia vas found despite the development of severe pyelonephritis.

Serum Hemagglutinins Hemagglutination tests were done on the blood obtained from all animals when thevx were killed. No hemagglutinins against E coli lipo- polvsaccharide were found until 8 days, when 3 of 16 rats infected with E coli 0:111 developed antibodv. In two the titer was 1:16 and in the third 1: 64. By 15 days serum hemagglutinins were found in all infected rats, in titers ranging from 1:4 to 1:64 (mean 1: 23). At 28 days, when infection had disappeared from all pvelonephritic kidneys, antibody wvas no longer found in the blood of 50! of the animals and in lower titers (mean 1: 10). In other words the humoral antibodv response, though transient, coincided with recoverv from infection. Discussion These studies establish for the first time that E coli can ascend the ureter and produce severe suppurative pvelonephritis without concomitant bacteremia. For this to occur, however, partial and temporarv obstruction of the ureter is necessary, since kidneys on the nonligated side never developed pyelonephritis or any, other abnormalitv even though they contained significant numbers of bacteria at various stages of the experiment. In the absence of inflammation, the consistent find- ing of E coli in the normal kidney probably represents continuing reflux of bacteria from the bladder rather than infection. Even highly virulent E coli, such as the 06 serotype used in these studies, cannot produce pyelonephritis without an element of partial ureteral obstruction. This organism virtually destroyed the kidney in the face of temporarv incomplete ureteral obstruction, but produced no disease in the opposite unobstructed side. Previous studies on "ascending" renal infection have produced pyelonephritis without obstruction but, as noted earlier, these Vol. 74, No. 2 RETROGRADE PYELONEPHRITIS 351 February 1974 were showkn by Fierer 5 to be the result of concomitant bacteremia in animals given such a large bladder infusion that pyelovenous reflux occurred. One of the remarkable features of this model is the low inoculum required to produce suppurative infection of the kidnev. Injection of only 50 to 100 bacteria in a .4-ml volume produced fulminant pyelonephritis with E coli 06 and severe pvelonephritis with E coli 0:111. Since human infection of the bladder is also initiated bv introduction of relatively few bacteria, since partial obstruction is an important accompaniment of human pyelonephritis, and since E coli is by far the leading cause of pyelonephritis in patients, this is a realistic model for studving immunity and immunization in this disease. Once the bacteria have been delivered to the pelvis of the kidney, their mode of entrance to the parenchnma is not known. One pathway that has been considered is pyelovenous, but this implies trauma to the pelvic epithelium and direct entrance into the blood stream, and con- sequently positive blood cultures."1 A second possibility is pvelolym- phatic flow, and there is considerable evidence in favor of this idea. Corriere 12 found that 24 hours after refluxing carbon particles, they were visible at all the fornices, but spared the papilla. Late spread of particles followed the renal lyvmphatics. Thus its earlv distribution mirrors that of our experiment at 20 hours. Other authors believe that pyelolymphatic backflow is the normal escape mechanism from the pelvis when the pressure rises. In support of this they inoculated labeled gold and albumin into the pelvis when obstructed and later isolated these substances from the thoracic duct, but not from the blood.1l Experiments in the dog, where E coli were inoculated into the obstructed renal pelvis, showed that they could be isolated from the lymph node betveen the hilum and the cisterna chyli but not from the blood."3 Others believe that normal pelvic epithelium is impermeable even to proteins but that rising intrapelxic pressure distorts the epithelium and creates "artificial mucosal openings."14 Certainly the fornices, which during water deprivation are lined bv simple squamous epithelium, would seem especially susceptible. The continuing damage at the superior fornix could be explained by the curious pumping action of the pelvis, vhereby urine coming out of the papilla is immediatelv pumped up around the papilla again.'5 The subsequent spread of the infection after 20 hours fits well with the current theories of the effect of urea on bacteria and the relationship of tissue osmolality to infection. WN'ater-deprived animals develop a very hypertonic papilla chiefly due to urea.'5 There is a gradient of 352 BROOKS ET AL American Journal of Pathology diminishing tonicitv toward the cortex. The osmolalitv of the tip of the papilla equals that of the pelvic urine.'6 Both Kaye ' and Schlegel 18 have shown the powverful antibacterial effect of urea, and Schlegel has protected against lower urinary tract infection bv giving animals high concentrations of urea in their drinking water.19 This is probably why the papilla is so often spared in our experiments. One also wonders whether the center of the medulla has a higher urea concentration than the periphery as the infection seems worse in the peripherv of the upper part of the papilla. The disease rarely spreads from the anterior or posterior fornices,20 which extend into the outer zone of the outer medulla. Extension from the superior fornices is the most common, and this lies in the inner zone of the outer medulla which is more hvpertonic, and probablv offers optimum conditions for early spread.2' Although not hypertonic enough to prevent migration of leukocytes into the area, it may inhibit phagoev- tosis.22 Subsequent sterilization of the lesions is associated with decreasing numbers of PMNs and increasing numbers of lvmphocytes. At 15 days only 29% of the ligated kidneys were still infected, and 80% of these had bacteriuria and prostatic abscesses. This seems to reemphasize the work of Friedlander, vho stressed the importance of prostatic lesions in pyelonephritis persisting beyond the acute stage.'0 The sterilization of the lesions was also associated with the appearance of antibody against E coli in the blood and suggests that humoral antibody may contribute to recovery from active pyelonephritis. Accordingly the rats were actively immunized to produce humoral antibody for the prevention of retrograde E coli pyelonephritis. These experiments are described in the next paper in this series." The human urinary pathogen 06 (Riffle) gave rise to much more severe disease than E coli 0:111. This mav be due to its hemolysin, but we were unable to repeat Fried's24 work with hemolytic strains of E coli. It is also resistant to the bactericidal power of rat serum, whereas E coli 0:111 is sensitive to the serum bactericidal activity. This difference in susceptibility to killing may be an important determinant of pathogenicitv for the kidney.

References 1. Keane WF, Freedman LR: Experimental pyelonephritis. XIV. Pyelonephritis in normal mice produced by inoculation of E. coli into the bladder lumen during water diuresis. Yale J Biol MIed 40:231-239, 1967 2. Montgomerie JG, Kalmanson GM, Hubert EG, Guze LB: Pyelonephritis. Vol. 74, No. 2 RETROGRADE PYELONEPHRITIS 353 February 1974

XIVI. Effect of immunization on experimental Escherichia coli pyelonephritis. Infect Immun 6:330-334, 1972 3. Heptinstall RH: Experimental pyelonephritis: bacteriological and morpho- logical studies on the ascending route of infection in the rat. Nephron 1:7-3- 92, 1964 4. Arana JA, Kozij VM, Jackson GG: Retrograde E. coli urinarv tract infection in rats: pathologic and bacteriologic characteristics of ascending pyelonephritis. Arch Pathol 78:558-567, 1964 5. Fierer J, Talner L, Braude AI: Bacteremia in the pathogenesis of retrograde pyelonephritis in the rat. Am J Pathol 64:443-45, 1971 6. Enta T, Lockey SD, Reed CE: A rapid safe technique for repeated blood collection from small laboratory animals: the farmer's wife method. Proc Soc Exp Biol MIed 127:136-137, 1968 7. Shapiro AP, Braude Al, Siemienski J: Hematogenous pyelonephritis in rats. IV. Relationship of bacterial species to the pathogenesis and sequelae of chronic pyelonephritis. J Chin Invest 38:1228-1240, 1959 8. Noll H, Braude AI: The preparation and biological properties of a chemi- cally modified Escherichia coli endotoxin of high immunogenic potency and low toxicity. J Chin Invest 40:1935-1951, 1961 9. Beer H, Staehelin T, Douglas H, Braude AI: Relationship between particle size and biological activity of E. coli boivin endotoxin. J Clin Invest 44:592- 602, 1965 10. Friedlander AM, Braude AI: Experimental prostatitis: its relationship to pyelonephritis. J Infect Dis 126:645-651, 1972 11. Chisholn GD, Rivero OR, Calnan JS: The renal pelvis in ureteral obstruction. Br J Surg 54:1023-1026, 1967 12. Corriere JN Jr, Murphy JJ: Vesicoureteral reflux and the intrarenal lvm- phatic system of the rat. Invest Urol 4:556-569, 1967 13. Murphy JJ, Myint MIK, Rattner WH, Klaus R, Shallow J: The l-mphatic system of the kidney. J Urol 80:1-6, 1958 14. Heney NM, O'.Morchoe PJ, O'Morchoe CC: The renal lymphatic system during obstructed urine flow. J Urol 106:455-462, 1971 15. Schmidt-Nielsen B: The renal excretion of nitrogen-containing metabolites, Urea, Vol 1. 1, Progress in Nephrology, Proceedings of the Svmposium of the Gesellschaft fur Nephrologie, Switzerland, 1967. Heidelberg, Springer-Verlag, 1969, pp 26-31 16. Valtin H: Sequestration of urea and non urea solutes in the renal tissue of rats with hereditary hypothalamic diabetes insipidus: effects of vasopressin and dehydration on the counter-current mechanism. J Chin Invest 45:337-345, 1966 17. Kaye D: Antibacterial activity of human urine. J Chin Invest 47:2374-2390, 1968 18. Schlegel JV, Cuellar J, O'Dell RM: Bactericidal effect of urea. J Urol 86: 819-822, 1961 19. O-Dell RNI, Brazil WO, Schlegel JU: Effectiveness of urea in prophylaxis of experimentally induced bacteriuria in rats. J Urol 97:145-146, 1967 20. Pfeiffer EW: Comparative anatomical observations of the mammalian renal pelvis and medulla. J Anat 102:321-331, 1968 354 BROOKS ET AL American Journal of Pathology

21. Andriole VT: Water, acidosis, and experimental pyelonephritis. J Clin Invest 49:21-30, 1970 22. Chemew I, Braude AI: Depression of phagocytosis by solutes in concentrations found in the kidney and urine. J Clin Invest 41:1945-1953, 1962 23. Brooks SJD, Lyons JMI, Braude AI: Immunization against retrograde pyelonephritis. II. Prevention of retrograde Escherichia coli pyelonephritis with vac- cines. Am J Pathol 74:359-364, 1974 24. Fried FA, Wong RJ: Etiology of pyelonephritis: significance of hemolytic Escherichia coli. J Urol 103:718-721, 1970 Fig 1-Extemal (top) and cut (bottom) surface of kidney on the ligated side 3 days after infusion with E coli 06 Riffle, showing multiple abscesses. This is indistinguishable from 0:111 infection at 3 days. '9 a~~~'I 4 -." *l I

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Fig 3-Typical section of infected kidney at 3 days showing infiltrate of the upper papilla and sparing of the papillary tip and center (H&E, X 60). B

Fig 4-Intravenous pyelograms showing free flow of dye past the ligature a few minutes postoperatively (A) and at 3 days immediately before sacrifice (B).