Pyelonephritis: The relationship between infection, renal scarring, and antimicrobial therapy

Kidney International, Vol. 19 (1981), pp. 654—662

Pyelonephritis: The relationship between infection, renal scarring, and antimicrobial therapy THOMAS MILLER and SUSAN PHILLIPS Department of Medicine, Auckland Hospital, Auckland, New Zealand

Pyelonephritis: The relationship between infection, renal scar-

reins avaient été infectCs ont ete traités apres un intervalle libre

ring, and antimicrobial therapy. We studied the relationship

variable et l'effet du traitement antimicrobien sur les aspects bactCriologique, anatomo-pathologique, et histologique de l'af-

between infection, renal scarring, and antimicrobial therapy in pyelonephritis by an experimental model of the disease. Our specific concern was to identify those aspects of the acute phase of infection associated with the induction of the inflammatory response. Our hypothesis was that the degree of the inflamma-

fection a éte déterminé. Les évènements précoces en relation avec l'invasion bactérienne du rein sont des determinants importants mais l'hypothèse doit être étendue pour rendre compte du

fait que les agents antimicrobiens administrés aprés que le

tory response and the extent of the subsequent lesion in the kidney depended on the rate at which a critical number of

nombre de bactéries alt atteint un plateau réduisent encore de

microorganisms was reached in the kidney. We proposed that the rapid increase in bacterial numbers in the kidney was the stimulus initiating the inflammatory response and not the total number of microorganisms in the kidney. In the experimental investigation, we treated animals with induced renal infection at varying intervals after the establishment of infection, and we

impliqués dans Ia genése des lesions pydlonephrétiques. Le

determined the effect of antimicrobial therapy on the bacteriologic, gross, and histopathologic features of the disease. The early

fibrose rénale peut Ctre empêchée ou significativement diminuée par un traitement antimicrobien rapide.

facon importante les lesions rénales. Deux facteurs semblent etre

premier est La vitesse d'installation de Ia population bactdrienne

dans un organe préalablement sterile. Un facteur additionnel important, cependant, est Ic nombre total de micro-organismes

du rein jusqu'au 4 jours aprCs Ic debut. Ces résultats ont une implication clinique dans La mesure ofl ils dCmontrent que Ia

events relating to the bacterial invasion of the kidney were important determinants but the hypothesis needed extending to account for the fact that antimicrobial agents, administered after bacterial numbers had reached a plateau, still reduced markedly the damage to the kidney. Two factors seem to be involved in the

An understanding of the relationship between the establishment of infection in the kidney, the development of renal scarring, and the effect of antimicrobial agents on the course of infection is essential if the basic biology of the disease is to be understood and its therapeutic management improved. Our reason for undertaking these experiments was to test an hypothesis that the degree of the inflammatory response and the extent of the subsequent lesion in the kidney depended on the rate at which a critical number of microorganisms was reached in

genesis of the pyelonephritic lesion. The first is the rate of acquisition of a bacterial population by a previously sterile organ, but an additional and important component is the total

number of microorganisms in the kidney up to 4 days after challenge. Clinically, the results are relevant in that they demon-

strate that renal scarring can be prevented or significantly reduced by prompt antimicrobial therapy. Pyélonéphrite: Relation entre l'infection, Ia fibrose rnale, et le traitement anti-microbien. La relation entre l'infection, la fibrose rdnale, et le traitement antimicrobien au cours de Ia pyélonéph-

rite a ete étudiée au moyen d'un modèle exp&imental. Notre

the kidney and not on the absolute number of

objectif spécifique était d'identifier les aspects de Ia phase aigue de l'infection associés a l'induction de Ia reponse inflammatoire. Notre hypothese était que le degre de Ia réponse inflammatoire et l'Ctendue de La lesion ultérieure rénale dependent de la vitesse avec laquelle un nombre critique de micro-organismes est atteint dans le rein. L'augmentation rapide du nombre de bactéries dans Ic rein a eté proposee comme pouvant être le stimulus qui initie la

microorganisms in the kidney. This concept arose from our observations that the direct introduction

of microorganisms into the kidney of otherwise unmanipulated animals led to rapid bacterial growth

and the development of gross, but circumscribed lesions [1]. On the other hand, it has been possible to demonstrate in two unrelated experiments that

reponse inflammatoire plutôt que Ic nombre total de microorganismes. Dans l'dtude experimentale, des animaux dont les

the number of microorganisms in the kidney did not necessarily reflect the pathologic status of the kidney. Thus, pathologic lesions do not develop in the kidneys of animals with acquiescent infection [2], although bacterial levels are comparable with those in kidneys with gross lesions. Similarly, inflamma-

Received for publication April 28, 1980 and in revised form August 18, 1980

0085-2538/81/0019-0654 $01.80

© 1981 by the International Society of Nephrology

654

Pyelonephritis: Infection, scarring, and therapy

tory renal lesions do not develop when animals are

treated with thiamphenicol if treatment is commenced at the time of challenge [31. In the latter experiments, the number of organisms in the kidney

of treated animals with no visible lesions were similar to those found in the kidneys of untreated pyelonephritic animals. In both situations, maximum bacterial numbers in the kidney were reached

over a period of weeks in contrast to the rapid acquisition of an infectious mass that occurred when microorganisms were introduced directly into the kidney.

Our thesis, therefore, was that the rapid expansion of bacterial numbers in the kidney was the stimulus that initiated the inflammatory response,

in contrast to the effect of a similar number of microorganisms acquired at a slower rate and subjected to the moderating influence of specific immune defense mechanisms. In the present experiments, animals with renal infection were treated with antimicrobial agents before and after the establishment of infection, and the effect of therapy on the bacteriologic, gross pathologic, and histopatho-

logic features of the disease was determined. The

results have shown that the number of bacteria maintained in the kidney during the first 4 days after

challenge, rather than the rate of bacterial replication during the acute phase of infection, was the critical factor initiating the inflammatory response. Methods Animal strain. Female Dark Agouti rats from an

inbred strain and weighing about 200 g each were used. Induction of renal infection. Pyelonephritis was induced by the inoculation of Escherichia coil 075 into the surgically exposed kidney. We used 10 l of a saline suspension of E. coli diluted to contain 3.2 x i03 microorganisms. Details of the surgical procedure have been given previously [4]. Antimicrobial therapy. Treatment of animals with

655

lothin, 32 p.g/ml) [5]. The maximum period during which animals with renal infection were treated was

I week. The rationale for the selection of this regimen, as opposed to a longer interval, was based on exploratory experiments showing that the conclusions reached after I week of treatment were not altered by prolonged therapy. Bacterial content of renal tissue. Euthanasia was

carried out by carbon dioxide inhalation, and the kidneys were removed with a sterile technique. Nutrient agar pour plates were made of serial tenfold dilutions of homogenized kidneys to obtain the bacterial count per gram of wet renal tissue. Minimum inhibitory concentration of the antimicrobial agents to E. coli 075. The MIC was determined by standard procedures. The following figures were obtained: cephalothin, 1.6 .g/ml; carben-

icillin, 1.6 g/m1; nitrofurantoin, 6.25 p.g/ml; ampicillin, 1.6 g/m1. Histologic assessment of renal damage. Quanti-

tative assessments of both the degree of renal

damage and the composition of the cellular infiltrate were made from sections stained with hematoxylin and eosin (H & E). A kidney profile was reproduced on paper, and the degree of renal damage, estimated

microscopically at a magnification of x 40, was shaded in on the profile. The profile was weighed and reweighed after the shaded area had been cut

out. The area of the lesion was expressed as a percentage of the total area of the kidney section. A quantitative assessment of the cellular components of the pyelonephritic lesion was made with the aid of a graticule in the eyepiece of the microscope. The number of cells per unit area (0.24 mm2) in the lesion was determined and differentiated into polymorphonuclear leukocytes, mononuclear cells,

and nuclear remnants derived from the renal and tubular epithelial cells.

Quantitative evaluation of gross pathologic

of at least eight times the minimum inhibitory concentration were produced. Confirmation was

changes. Quantitation of the degree of gross renal pathology was made with the aid of a kidney profile stamp that was divided into ten equal areas representing the surface area of the kidney. Scarred or infected areas were then shaded in on the kidney profile and expressed as a percentage of the total surface area of the kidney. Tissue processing. Renal tissue was placed directly into a 0.1% solution of cetylpyridinium chloride in 10% formaldehyde. After routine processing and cutting, sections were stained with H & E.

either by direct experimentation (ampicillin, peak serum concentration of 31 g/ml; carbenicillin, 75 pg/ml) or from evidence in the literature (cepha-

carried out with the Wilcoxon sum of Ranks test for nonparametric data using a PDP II computer.

pyelonephritis was carried out with ampicillin (Pen-

britin), 75 mg/kg i.m.; cephalothin (Keflin), 100 mg/kg i.m.; carbenicillin (Pyopen), 150 mg/kg i.p.;

and nitrofurantoin (Furadantin), 100 mg/kg p.o. These doses were all administered every 24 hours and according to the protocols detailed in the text for individual experiments. Serum concentrations

Statistical analysis. Analysis of the data was

656

Miller and Phillips

respectively, for the 2.5-hr prechallenge and 16-hr postchallenge data).

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Renal infection was established in four groups, each of 10 animals. One group remained untreated and served as a control for the experiment. The second group was treated with carbenicillin (150 mg/kg), the third with nitrofurantoin (100 mg/kg), and the

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Fig. 1. Bacterial numbers in the kidneys after the direct inoculation of3.2 x 1J E. coli into the kidneys. Each point is the mean

of the bacterial counts from the kidneys of 6 animals.

Results

Bacterial replication of the kidney. E. co/i (3.2 X 10) were introduced directly into the kidney. The number of microorganisms decreased during the first 2 hours, but during the subsequent 48 hours

fourth group with cephalothin (100 mg/kg). The animals receiving antimicrobial agents were treated

daily over a 7-day period commencing 24 hours after challenge. All groups were examined 11 days after challenge, that is, 4 days after the last dose of antibiotic. As with other experiments, the bacteriologic status of the kidney of the treated animals at the time of sacrifice did not differ from the controls (Fig. 3). Antimicrobial therapy, even when delayed

as long as 24 hours after challenge, still reduced significantly the development of gross lesions in the kidney for carbenicillin (P < 0.01), nitrofurantoin (P

Thereafter, microbial numbers in the kidney re-

<0.02), and cephalothin (P < 0.01). Antimicrobial therapy: Time of commencement

mained largely unaltered over a period of 10 days,

as a factor affecting the development of gross

rapid replication led to a 1000-fold increase (Fig. 1).

and other experiments, not reported here, have shown that this level of infection is maintained for several months. Effect of antimicrobial agents on scar formation. Experiments were carried out to determine whether the effect of thiamphenicol on renal infection, that

lesions. It was apparent that antimicrobial therapy, commenced at a time when bacterial numbers had reached a plateau, could still influence the development of gross pathologic lesions. This was so even though treatment commenced at this stage could not interfere with the phase of rapid bacterial repli-

is, the prevention of scar formation without an

cation in the kidney (Fig. 1). This observation

apparent reduction in bacterial numbers, could be observed with other antimicrobial agents. (a) Ampicillin. Renal infection was induced in

suggested that the original concept of pathologic changes in the kidney being initiated during the

three groups of 14 animals, one of which was untreated and served as a control. The second group was treated with a single dose of ampicillin (75 mg/kg) 2.5 hours prior to challenge. In the third group, the initial dose of ampicillin was not given until 16 hours after the establishment of infection

but was continued for 4 days. All animals were sacrificed 10 days after challenge, and the gross pathologic and bacteriologic status of the kidney was determined. The results (Fig. 2) have con-

phase of rapid replication in the kidney needed to be modified. In the following experiments, the timing of commencement of antimicrobial therapy in relation to the gross pathologic lesion and the histopath-

ologic and bacteriologic status of the kidney was studied systematically with carbenicillin as a representative antimicrobial agent. Renal infection was induced in 12 groups, each of 10 to 15 animals. Six

groups were untreated and served as controls for

the experimental series, and the remaining six groups were treated with 150 mg/kg of carbenicillin

firmed those obtained with thiamphenicol and have

daily for 7 days. Treatment of groups 1 through 6

shown that antimicrobial therapy from 2.5 hours before challenge to 16 hours after challenge may reduce significantly the development of gross lesions in the kidney without affecting the bacteri-

commenced 8 hours and 1, 2, 3, 4, and 7 days, respectively, after infection. Four days after the

ologic status of the kidney (P < 0.01 and 0.05,

pathologic renal damage was made, and the compo-

final administration of the antibiotic, the animals were killed. An assessment of the gross and histo-

657

Pyelonephritis: Infection, scarring, and therapy

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the gross lesions on the surface of the kidney. This was evident even when the initiation of treatment was delayed as late as 4 days after the establishment of infection (Table 1). The effect of antimicrobial

therapy in reducing the size of the lesion was

Miller and Phillips

658

Table 1. Effect on the gross pathology of the renal lesion when

commencement of antimicrobial therapy is delayed Carbenicillin-treated group (150 mg/kg/day for 7 days) Gross pathology of nontreated controlsa % 34.0 46.0 28.0 32.0 31.0 22.0

of kidneys at autopsy was then determined. Al-

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aDegree of lesion formation is expressed as a percentage of the surface area of the kidney. Animals were sacrificed 4 days after SEM. the final dose of antibiotic. Values are the means "Time after animals challenged that therapy was started 'Degree of significance is for treated animals compared with untreated controls, using the Wilcoxon sum of Ranks analysis.

tained when treatment of further groups of animals was commenced ito 7 days after challenge (Fig. 5). The results were in contrast to the reduction in the gross and histopathologic changes observed in the kidneys of animals when antimicrobial therapy was commenced within 4 days of challenge (see Tables 1 and 2). Effect of antimicrobial therapy on bacterial num-

bers. The data presented so far have shown that antimicrobial therapy can affect the gross and histopathologic changes in the kidney but that the bacteriologic status in the treated and nontreated groups

Table 2. Effect on histopathologic damage when commencement of antimicrobial therapy is delayed

at the time of sacrifice may be similar. We next considered the possibility that the bacterial numbers in the kidney may be suppressed initially by antimicrobial therapy, but then increase, so that at the time of sacrifice the bacteriologic status of the

Carbenicillin-treated groupb (150 mg/kg/day for 7 days)

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The number of microorganisms in the kidney at varying times after treatment was therefore determined to establish the bacteriologic status of the kidneys during therapy. In the first group in which therapy commenced 8 hours after challenge (Fig. 6a), bacterial numbers in the untreated group in-

aHistopathologic damage is expressed as percentage of surface SEM. area of sectioned kidney. Values are the means bAnimals were sacrificed 4 days after the final administration of antibiotic. Gross pathology is expressed as a percentage of the surface area of the kidney. cTime after animals challenged that antibiotic therapy started dDegree of significance is for treated animals compared with untreated controls, using Wilcoxon sum of Ranks analysis.

creased tenfold over the subsequent 16-hour period but then remained at a constant level. In the treated group, antimicrobial therapy prevented an expansion of bacterial numbers in the kidney, and significant differences between the treated and untreated groups were demonstrable for all the time periods

particularly notable when treatment commenced

in renal damage could be demonstrated when anti-

teau at the time antimicrobial therapy was initiated at 24 hours. Treatment with carbenicillin reduced the number of microorganisms in the kidney up to 7 days after challenge. Similar results were obtained when therapy commenced 48 hours after challenge when again highly significant reductions in bacterial

microbial therapy was begun within 4 days of

numbers were achieved up to 7 days after the

challenge (Table 2). (c) Cellular infiltrate. The density of the cellular

induction of infection (Fig. 6c). When the initiation

infiltrate per unit area (approx. 25 high-power

challenge, no significant reduction of bacterial num-

fields) was scored. The data obtained was independent of the total area of the kidney affected. Antimi-

bers was found (Fig. 6d). These experiments have shown that antimicrobial therapy, initiated within 3

within 24 hours of infection. (b) Quantitative histopathologic assessment. The

results obtained by histopathologic examination agreed with the gross assessment, and a reduction

studied. In the second group (Fig. 6b), bacterial numbers in the kidney had already reached a pla-

of treatment was delayed up to 72 hours after

659

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carbenicillin (150 mg/kg) daily for 7 days. The interval between challenge and the commencement of therapy is indicated for each group: 8 hr, I day, etc. Autopsies were carried out 4 days after the final dose of the antibiotic had been given. PMN denotes polymorphonuclear

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days of challenge, temporarily reduced the number of microorganisms found in the kidney during the early phase of infection. No significant differences

modified by the use of antimicrobial agents. The experiments show that renal lesions develop as a

in bacterial numbers were found when the commencement of antimicrobial therapy was delayed beyond that time.

sustained concentration of metabolically active

Discussion

In this study, we investigated the hypothesis that the rate of bacterial replication in the kidney is the critical determinant initiating the inflammatory re-

sponse. As a result of the experiments, we have established the relationship between the infecting microorganisms and the development of renal scar-

ring, as well as the conditions under which the pathogenesis of the pyelonephritic lesion can be

result of the inflammatory response of the host to a microorganisms resulting from an acute infection of the kidney. This destructive inflammatory response of the host needs to be distinguished from the hostparasite relationship found in acquiescent infection, in which, characteristically, a high bacterial load in

the kidney does not stimulate an inflammatory response. Furthermore, we found that the adminis-

tration of antimicrobial agents up to 4 days after challenge, at a time when bacterial numbers in the kidney had reached a peak, still markedly reduced the degree of gross and histopathologic damage. But, neither the lesion nor the bacteriologic status

Miller and Phillips

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of the kidney was affected by antimicrobial agents administered after infection had been established for more than 4 days. Thus, our original thesis that the early events relating to the bacterial invasion of

the kidney were the important determinants was largely correct but needed extending to account for

the fact that antimicrobial agents, administered after bacterial numbers had reached a plateau, still markedly reduced the damage to the kidney.

A number of the concepts developed in the present experiments are remarkably similar to those discussed by Miles [61 24 years ago relating to the establishment of infection in localized skin lesions. Our idea of the pathogenesis of the lesion in pyelonephritis is that the inflammatory response initiating the damage is associated with the early stages of infection but that two factors are involved. The first is the rate of acquisition of a bacterial population by

a previously sterile organ, but the total number of microorganisms in the kidney up to 4 days after challenge is an additional and important component. The latter factor most probably relates to the level of the chemotactic factors produced by the pathogens. In these experiments, the rate of bacterial growth in the kidney was first determined. Rapid replication of microorganisms in the kidney occurred, and

a thousandfold increase in bacterial numbers was demonstrable during the first 48 hours after challenge. The hypothesis that the rapid increase in bacterial numbers stimulated the inflammatory response arose from a study of the circumstances in

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which large numbers of microorganisms, usually associated with an active disease, could be present in the kidney without inducing an inflammatory response. These were the acquiescently infected kidney [2] and the bacteriologic status of the kidneys of animals treated with thiamphenicol before the induction of infection [3]. The feature common to both these situations was that the microbial load in the kidney was acquired slowly in contrast to the

course of the acute infection. As a result, it was postulated, the host's response to infection was modified, and a tissue-destructive cellular immune response was not mounted. The results of the present experiments support previous observations of the effect of thiamphenicol on renal infection, that is, that the administration of an antibiotic at the time or shortly after the induction of experimental renal infection prevented the development of renal lesions without altering the bacteriologic status of the kidney in the long term.

This effect could be demonstrated with several other antimicrobial agents, and animals treated with ampicillin, carbenicillin, nitrofurantoin, and cephalothin all showed a significant reduction in the gross

and histopathologic changes in the kidney when antimicrobial therapy was commenced within 4 days of challenge. One observation requiring explanation was that the bacteriologic status of nontreated animals with pyelonephritis and animals treated within 4 days of

challenge was similar at the commencement of antimicrobial therapy and at sacrifice 10 to 14 days

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was commenced from 8 hours to 3 days after challenge. The object was to determine the intermediate bacteriologic status between commencement of antibiotic treatment and autopsy. The times shown for each group signify how long after challenge the animals were sacrificed.

after challenge. The lesions in the treated group, however, were markedly reduced compared with nontreated controls, despite an apparently similar bacteriologic status. We investigated the possibility that in the interval between therapy and sacrifice, the numbers of microorganisms in the kidney were

initially reduced by antimicrobial treatment, but later increased, so that at sacrifice (10 to 14 days after challenge) no difference in the bacteriologic status of the kidneys from treated and untreated groups was apparent. Evidence from the experiments (Fig. 6, a—d) supported this suggestion, and significant reductions in bacterial numbers in the kidney were achieved during antimicrobial therapy. Thus, the hypothesis that the early phase of tissue

invasion by microorganisms was the critical determinant in the pathogenesis of the lesion was largely correct although it is now evident that antimicrobial therapy commenced within 4 days of challenge may

still reduce the bacterial numbers sufficiently to modify the degree of infection-induced inflammatory response. Studies of the effectiveness of antimicrobial therapy in experimentally induced renal infection have been undertaken by a number of investigators, but the majority of these have been concerned with the effect of therapy on established lesions [7, 8]. The general experience, however, has been similar to our own in that once lesions have been established in the kidney the natural history of the lesion is not

Miller and Phillips

662

affected by altering the bacteriologic status of the kidney. Our specific concern in these experiments was to

identify those aspects of the acute phase of infec-

ance of Mrs. Denise Wilson, who prepared the tissue for histopathologic examination.

Reprint requests to Dr. T. Miller, Department of Medicine, Auckland Hospital, Park Road, Auckland, New Zealand

tion specifically associated with the induction of the

inflammatory response. The evidence from the present experiments strongly suggests that the number of invading organisms in the renal parenchyma and, presumably, their metabolic activity during the early stage of infection are the critical determinants. Similar conclusions have been reached by Glauser,

Lyons, and Braude 191. In their opinion also, the response to acute infection rather than the continued presence of microorganisms in the kidney is the critical factor initiating renal damage. These results are clinically relevant and demonstrate that prompt

and effective antimicrobial therapy of acute renal infection can prevent or significantly reduce the development of renal scarring. Such an awareness could be important in the treatment of recurrent acute infections associated with obstruction and other anatomical defects of the urinary tract.

References 1. MILLER TE, LAYZELL D, STEWART E: Experimental pyelo-

nephritis: The effect of chronic active pyelonephritis on renal function. Kidney mt 9:23—29, 1976 2. MILLER TE, NORTH D, BURNHAM S: Acquiescent renal infection. Kidney mt 7:413-421, 1975 3. MILLER TE, BURNHAM 5, NORTH JDK: Immunological enhancement in the pathogenesis of pyelonephritis. C/in Exp Immunol 24:336—345, 1976 4. MILLER TE, ROBINSON KB: Experimental pyelonephritis: A

new method for inducing pyelonephritis in the rat. J Infect Dis 127:307—310, 1973 5. FABRE J, BLANCHARD P, RUDHARDT M: Pharmacokinetics

of ampicillin, cephalothin and doxycycline in various tissues of the rat. Chemotherapy 23:129—141, 1977 6. MILES AA: Non-specific defense reactions in bacterial infections. Ann NYAcad Sci 66:356—369, 1956 7. GLASSOCK RJ, KALMANSON GM, GUZE LB: Pyelonephritis

XVIII. Effect of treatment on the pathology of enterococcal pyelonephritis in the rat. Am J Pathol 76:49—62, 1974 8. HUNTER BW, SOUDALL LL, SANFORD JP: Antibiotic ther-

apy in established chronic experimental Proteus mirabilis pyelonephritis. Antimicrob Agents Chemother 3:608—612,

Acknowledgments

This work has been supported by the Medical Research Council of New Zealand. We acknowledge the technical assist-

1963

9. GLAUSER MP, LYONS JM, BRAUDE Al: Prevention of chron-

ic experimental pyelonephritis by suppression of acute suppuration. J C/in Invest 61:403-407, 1978