effect of indomethacin that is related to the renal response to infection. This was possibly due .... of kidney or cortex or per milliliter of bladder or pelvic urine were ...
INFECTION AND IMMUNITY, May 1983, p. 529-533 0019-9567/83/050529-05$02.00/0 Copyright C 1983, American Society for Microbiology
Vol. 40, No. 2
Effect of Indomethacin on the Incidence of Experimental Escherichia coli Pyelonephritis MICHEL P. GLAUSER,* PATRICK B. FRANCIOLI, JACQUES BILLE, MARC BONARD, AND PASCAL MEYLAN Division of Infectious Diseases, Department of Medicine, Centre Hospitalier Universitaire Vaudois, 1011
Lausanne, Switzerland Received 19 November 1982/Accepted 3 February 1983
In the presence of temporary obstruction (20 h), ascending Escherichia coli urinary infection leads to irreversible acute exudative pyelonephritis (AEP) in rats. The purpose of the present study was to investigate the early inflammatory events which take place in response to the presence of bacteria in the kidney parenchyma and lead to the development of AEP. Rats were given indomethacin before and during the obstructive phase of kidney infection and were sacrificed at different times thereafter. Although renal infection (as defined by bacterial counts) was equally frequent (76%) and severe in indomethacin-treated and control rats sacrificed at the end of the obstructive period, it was found that the incidence of AEP (as defined by the inflammatory response of the kidney elicited by bacteria) 2 days after removal of the obstruction was significantly reduced from 74% in controls given water to 48% in indomethacin-treated animals (P = 0.02). Rat kidneys without AEP had bacterial counts of 102/g. Since indomethacin apparently had no direct antibacterial activity against E. coli and no effect on urine osmolalities, it is likely that the reduction in the incidence of AEP and the concomitant eradication of bacteria after removal of the obstruction was due to an effect of indomethacin that is related to the renal response to infection. This was possibly due to decreased inflammation, as indicated by the fact that when *pyelonephritis developed in indomethacin-treated rats it was less severe than in controls. These results suggest that if inflammation can be mitigated when bacteria are present in the kidney during obstruction, the bacteria may be cleared spontaneously once the normal urinary flow is restored.
Renal involvement during ascending urinary tract infection in humans is believed to occur in 30 to 50% of patients (3, 16, 17). If renal infection, in the absence of obstruction or underlying kidney disease, causes permanent damage,
chronic pyelonephritis (i.e., renal interstitial damage and scarring due to infection) would be a leading cause of renal failure. On the contrary, chronic pyelonephritis is seldom found as a cause of end stage renal disease and, when it occurs, it is almost always associated with urinary tract abnormalities (14) or with other causes of renal damage (13). In animals, after the inoculation of bacteria in the urine, infection clears rapidly from the normal kidney without significant exudation. Furthermore, intravenous inoculation of Escherichia coli does not cause kidney lesions in the absence of ureteral ligation, previous kidney damage, or hydropenia (1, 9). The purpose of the present study was to investigate the mechanisms by which infection, i.e., the presence of bacteria, causes severe
kidney suppuration and damage after temporary obstruction but not in the absence of obstruction. It was found that indomethacin, an antiinflammatory agent without antibacterial activity, diminished the incidence of subsequent acute E. coli pyelonephritis when given during the obstructive phase of kidney infection by allowing a substantial number of animals to clear bacteria once the urinary flow was restored. MATERIALS AND METHODS Pyelonephritis production. Ascending unilateral pyelonephritis was produced in male Wistar rats weighing 200 to 225 g each (Madorin AG, Fullinsdorf, Switzerland) as previously described (2), with slight modification (7). Briefly, after water deprivation for 18 h, animals were anesthetized with pentobarbitalum natrium (Nembutal Abbott AG, Zug, Switzerland) injected intraperitoneally. The left ureter was exposed through a midline incision of the abdominal wall. At midureter level, a silk suture was passed through the flank into the peritoneal cavity, over the ureter, and out again to the left flank. The ligature was left loosely in place while bacteria were injected through the dome 529
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of the bladder into the lumen. One milliliter containing 2 x 103 E. coli 06 (Williams) (2) of an overnight culture in tryptone soya broth (Oxoid Ltd., Basingstoke, Hants, England) was slowly infused. Ureteral reflux consistently occurred when urine appeared at the external urethral meatus, and the remainder of the 1.0ml volume was passed externally through the urethra without causing further bladder distension. After the infusion, the two arms of the ureteral ligature were tied loosely and the abdominal wall was closed. Twentyfour hours later, the ligature was removed. This procedure produced ureteral obstruction, and severe unilateral acute exudative pyelonephritis subsequently developed in about 75% of control rats. Three days after operation, at the peak of the inflammatory process, the pyelonephritic kidneys were greatly enlarged and displayed numerous small abscesses over the cortex. In animals without macroscopic pyelonephritis, there was no kidney enlargement and the kidney homogenate and urine remained sterile or showed only a few colonies. Indomethacin treatment. Groups of rats were treated with indomethacin at 12 h before operation and every 12 h thereafter for a total of three to five doses, depending on the experiment. Indomethacin powder (Indocid; Merck Sharp & Dohme, Zurich, Switzerland) was suspended in water at a concentration of 0.4 mg/ml. One milliliter of the suspension was administered by a nasogastric tube (Argyle feeding tube; Sherwood Medical Industries, Petit Rechain, Belgium) in such a way that each rat received 2 mg/kg per dose. Controls received 1 ml of water. At the time of operation, the dose was administered before anesthesia.
Sacrifice of animals. The animals were sacrificed 24 72 h after operation. After terminal anesthesia, the abdomen was opened and the kidneys were removed aseptically, observed for gross pathological lesions, weighed, and homogenized. Specimens of bladder and pelvic urine were collected with a tuberculin syringe and a 27-gauge needle. In some experiments, the kidneys were cut in half; one half was used for culture and the other half was processed for histological examination. Tenfold dilutions of the homogenates were plated on MacConkey agar (Difco Laboratories, Detroit, Mich.) incubated overnight at 37°C before counting the number of CFU. In experiments in which the rats were sacrificed 24 h after operation (with the ureteral ligature still in place), a slice of renal cortex was taken before removing the left kidney to avoid bacterial contamination by pelvic urine. Kidneys and or
cortex pieces were then aseptically weighed, homogenized, and plated separately for bacterial numeration.
Evaluation of severity of pyelonephritis. Kidney weight provides a quantitative measure for the severity of pyelonephritis (2). During the acute phase of obstructive pyelonephritis, kidney weight increases in proportion to the inflammation and suppuration. To minimize the variation of kidney weights between animals, the ratio of the left kidney weight to the right kidney weight (L/R kidney weight ratio) was used. The ratio is 1 in normal rats (7). Effect of indomethacin on renal concentrating capacity. Since indomethacin in high doses (10 mg/kg) has been shown to reverse the early urinary concentrating defect observed during kidney infection in rats (12), the effect of lower doses (2 mg/kg, as used in the present experiments) on urine osmolality was determined. Groups of eight rats each were placed in metabolic cages and allowed food and water ad libitum, and the osmolalities of 24-h urine collections under paraffin oil were measured by freezing point depression (Roebling Osmometer; Onfochroma AG, Zug, Switzerland). Three days after containment in metabolic cages, the animals were dehydrated and given indomethacin or water by a nasogastric tube starting 12 h before operation. They were subsequently operated and treated in the same manner as were rats in other experiments. Urine osmolalities were measured until sacrifice for evaluation of pyelonephritis 3 days post-operatively. Statistical evaluation. The numbers of CFU per gram of kidney or cortex or per milliliter of bladder or pelvic urine were compared by Student's impaired t test. Statistical comparison of the incidence of acute pyelonephritis between groups of animals was performed by the chi-square method with Yates' correction.
RESULTS Effect of indomethacin treatment on AEP 3 days after operation. To investigate the effect of indomethacin on the incidence of acute pyelonephritis, we treated groups of rats with five doses of indomethacin at 12-h intervals; the first dose was given 12 h before operation, and the last dose was given 36 h after operation, i.e., 12 h after removal of the obstruction. The animals were sacrificed 3 days after operation, i.e., 36 h after the last dose of indomethacin. Table 1 shows the results in 88 operated rats in
TABLE 1. Incidence and severity of infection and of gross lesions (AEP) 3 days after onset of infection' Treatment group
No. of rats
AEP absent L/R kidney wt ratio
logl0 CFU/g
AEP present No. of rats
L/R kidney wt ratio
log10 C FU/g
No. of rats with AEP/ total (%)
31/42 (74)c 1.90 ± 0.3b 7.49 ± 0.6 31 1.32 ± 0.6 1.10 ± 0.2 11 Control 22/46 (48)c 1.56 ± 0.3b 7.62 ± 0.6 22 1.37 ± 0.6 1.15 ± 0.1 24 Indomethacin treated a The presence of AEP is visible by gross examination; the pyelonephritic kidneys are enlarged and covered by numerous small abscesses scattered over the cortex surface. Kidneys with sterile culture were counted as having 1 E. coli organism per g. Results are shown as mean ± standard deviation. loglo b p = 0.001. c P = 0.02.
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four different experiments that revealed similar findings. There was a one-third reduction in the incidence of gross acute exudative pyelonephritis (AEP) after indomethacin treatment (from 74% in controls to 48% in indomethacin-treated rats; P = 0.02). In addition to this reduction, less-severe pyelonephritis was observed in treated animals that developed the disease, as judged by the L/R kidney weight ratio. Histologically, the sections of kidney from both control and indomethacin-treated rats were observed blindly. Within pyelonephritic lesions, no obvious differences in the degree of polymorphonuclear leukocyte infiltration, edema, or residual hydronephrosis could be detected. The magnitudes of infection, as measured by bacterial counts in pyelonephritic kidneys of both control and indomethacin-treated rats, were similar (Table 1). Rats that had not developed pyelonephritis (macroscopically normal-appearing kidney) had a L/R kidney weight ratio that was slightly higher than normal values, owing to residual hydronephrosis (7). The culture of the kidney homogenate was sterile in most of the animals (17 of 24 indomethacin-treated rats and 9 of 11 control rats without macroscopical kidney lesions). The remaining kidneys had less than 102 E. coli organisms per g of homogenate, as compared with 107 E. coli organisms per g in kidneys from animals with overt pyelonephritis (Table 1). The results of these experiments, therefore, suggested that the incidence of E. coli AEP could be reduced by indomethacin given before, during, and after the obstructive phase of the disease. Effect of indomethacin treatment in rats sacrificed 24 h after operation and before removal of obstruction. In this model, obstruction for 18 to 24 h is necessary to produce AEP after bladder injection of the refluxing bacterial inoculum. The present experiments were performed to investigate the possibility that the diminished incidence of AEP 3 days after operation in
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indomethacin-treated animals was due to lesssevere obstruction during the first 24 h of the disease, owing to the antiinflammatory effect of the drug. Rats were treated with three indorr, thacin doses (the first given 12 h before operati.on, the second at the time of operation, and the third 12 h after operation) and were sacrificed 24 h after operation with the ligature around the left ureter still in place. At the time of sacrifice, 24 h after operation, the macroscopic appearances (pale, edematous, without visible abscess) of the left obstructed kidneys were similar in both controls and indomethacin-treated rats, and there was no difference between infected and noninfected kidneys. Upon microscopic examination, control and indomethacin-treated kidneys were similar, and there was little or no visible inflammation at this stage. In contrast, two groups of kidneys could be distinguished upon culture in both treated and control rats (Table 2). First, there was a majority (72 to 74%) of left kidneys with severe infection (.106 E. coli organisms per g in either the renal cortex or the whole-kidney homogenate). The second group of left kidneys had no significant infection (
