ROLE OF BACTERIAL UREASE IN EXPERIMENTAL PYELONEPHRITIS'

ABRAHAM I. BRAUDE AND JENNIE SIEMIENSKIDepartment of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania

Received for publication December 9, 1959

Infections of the kidney are produced bybacteria that seldom infect other organs. This factis evident from an analysis of the relativefrequency with which different bacterial speciesare isolated in infected urines. Studies summa-rized in table 1 disclosed that Escherichia coliwas the most frequent cause of urinary infectionand Proteus species are next. Then in diminishingorder there were Aerobacter aerogenes, Klebsiellapneumoniae, paracolon organisms, Pseudomonasaeruginosa, enterococci, and Alcaligenes faecalis.Although these bacteria are generally regarded asrelatively harmless in comparison to Staphylo-coccus aureus and Streptococcus pyogenes, thevirulent gram-positive pathogens were rarely thecause of urinary infection. It would seem, there-fore, that the conditions enabling bacteria toproliferate in the kidney and damage it areuniquely different from those in other organs.Beeson and Rowley (1959) have presented evi-dence that kidney tissue can interfere with thebactericidal action of serum and suggested thatthe intrarenal formation of ammonia inactivatesthe fourth component of complement. Since thekidney is also distinguished from all other organsby its high concentration of urea, it might bepossible to account for the susceptibility of thekidney to Proteus on the basis of its powerfulurease. The following studies were undertaken,therefore, to investigate the role of Proteus ureasein the pathogenesis of pyelonephritis in anattempt to understand how relatively innocuousbacteria can selectively attack the kidney.

MATERIALS AND METHODS

Pyelonephritis was established in rats byintracardiac inoculation of bacteria and massageof the kidney in the manner described previously(Braude, Shapiro, and Siemienski 1959). Lightrenal massage has been shown to increase thenumber of bacteria that localize in the kidney(Braude et al. 1959). Sections were stained with

1 This work was supported by a grant from theU. S. Public Health Service (H-3220).

hematox-ylin and eosin, and imprints of individualrenal cells were stained with Giemsa's stain.Imprints were made by touching the cut surfaceof the pyelonephritic kidney to microscope slides.

Urease activity was preserved in suspensionsof dead Proteus mirabilis by killing the bacteriawith acetone. The bacteria were grown intrypticase soy broth at 37 C for 24 hr, separatedfrom the original culture medium by centrifu-gation, and resuspended in 32 per cent acetone.Urease and catalase were preserved indefinitelyat 4 C.To study the infection of kidney epithelium in

tissue culture, monolayers of monkey kidneyepithelial cells were grown in test tubes for 5 daysin Hanks balanced salt solution containing 0.5per cent lactalbumin and 2 per cent serum. Onthe sixth day the cells were washed three timeswith 1-ml quantities of balanced salt solution.Infection of the tissue culture was then es-tablished by a modification of the method ofShepard (1959). A loopful of an 18-hr blood agarculture of either E. coli (Gray strain) or P.mirabilis (North strain) was inoculated into 50ml of a medium adjusted to pH 7.5 to 7.7 with7.5 per cent NaH2CO3 and composed of Hanksbalanced salt solution, 1 per cent of medium 199,and 20 per cent calf serum. These bacterialsuspensions possessed faint but equal turbiditiesand were free of clumped organisms. Suspensionsof E. coli and P. mirabilis were each divided intoportions and urea was added to give concen-trations of either 0.05, 0.1, 0.2, or 0.3 per cent.No urea was added to a final sample. The sampleswere each divided among multiple tubes of tissuecultures of monkey kidney epithelium. The effectof urea alone, in the absence of bacteria, wasstudied in control tubes of kidney epithelialcultures containing the same concentrations ofurea as the infected cultures. The tubes wereincubated in the stationary position at 37 C for6 hr.

After 6 hr the infection medium was removedand replaced with the streptomycin growth

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TABLE 1Etiology of urinary infections in 448 patients at the

Presbyterian Hospital in Pittsburgh*Relative Incidence

(% of TotalInfections)

1. Escherichia coli............. 30.52. Proteus species ............. 18.43. Aerobacter aerogenes ........ 16.34. Klebsiella pneumoniae. 10.15. Paracolon species........... 6.36. Pseudomonas aeruginosa 5.87. Enterococci ................ 4.08. Escherichiafreundii. 3.39. Alcaligenes faecalis ......... 2.2

10. Staphylococcus epidermidis.. 2.011. Staphylococcus aureus ....... 1.1

* All urines were collected aseptically bycatheter from women and midstream catch frommen and plate counts performed by the methodof Sanford et al. (1956). More than 10,000 bacteriaper ml of urine were present in each of the 448patients comprising this analysis.

medium after washing with 1-ml quantities ofHanks balanced salt solution. The streptomycingrowth medium was composed of Eagle's mediumprepared with 40 per cent pooled human serumand 50 ,ug streptomycin sulfate per ml. The pHwas 7.6 to 7.9. The purpose of the streptomycinwas to prevent extracellular growth of bacteria.In other tubes the extracellular infection wasallowed to proceed without the addition ofstreptomycin.The infected tissue cultures and the controls

were incubated (37 C) for variable periods, andthe cells examined microscopically at regularintervals before removal from the tubes. Enoughtubes were inoculated to permit frequentsampling for the purpose of following the courseof the infection. At serial time intervals the cellswere removed with a smooth platinum loop andspread on a glass slide where they were stainedwith Giemsa's stain. The tubes were also sampledat frequent intervals for measurement of pH in aBeckman pH meter.

RESULTS

Renal damage unique to Proteus infection andattributable to urease. In comparative studies ofpyelonephritis due to a variety of bacterialspecies, two features have been observed that are

unique to Proteus infections and are believed tobe related to the activity of their urease:

(1) Kidney stones:-These were present in 13of 59 rats (22 per cent) examined in various stagesof acute and chronic pyelonephritis (Braude et al.,1959; Shapiro, Braude, and Siemienski 1959).They ranged in size from small concretions ofgravel near the papillary tip to large, typicalstaghorn calculi filling the entire renal pelvis. Intheir earliest stages the crystals could be observedas mineral deposits at the tips of the renalpapillae and were reminiscent of those describedby Randall (1939) in human kidneys. On analysisby E. Prien they were shown to be composed ofMgNH4PO4, a salt that precipitates in thealkaline environment produced by the accumu-lation of ammonium after decomposition of ureaby Proteus urease. Stones have not been found innumerous rats infected with E. coli, P. aeruginosa,or enterococci; nor in uninfected rats used ascontrols in these experiments.

(2) Selective intracellular parasitism andnecrosis of renal tubules:-The earliest stages ofProteus infection of the kidney were character-ized by dense proliferation of bacteria withintubular epithelium where urea concentration ispresumed to be high. Colonies of P. mirabiliswere observed to localize in the tubular cellswithin 18 to 24 hr after inoculation (figures 1 and1A) and stained imprints of individual tubularcells disclosed that the colonies developed in thecytoplasm (figure 2, 2A, and 2B). Infections withstaphylococci and E. coli on the other hand, didnot appear to localize or multiply in tubularcells; instead, these bacteria were found outsidethe tubules and extracellularly (figures 3 and 4,and Braude, Shapiro, and Siemienski, 1955a).Growth of Proteus in tubular epithelium pro-

duced necrosis of the tubular cells. By 24 hr theseinjured tubules were found to be the center of afocal inflammatory reaction beginning with a fewpolymorphonuclear leukocytes and later com-posed of concentric masses of these cells (figure1A). This peritubular reaction increased toproduce the massive wedge-shaped cortico-pelvicinflammatory lesions that are characteristic ofProteus pyelonephritis (Braude et al., 1959). In30 per cent of kidneys, even the early lesionspreceding inflammation produced massive grosslesions. These pre-inflammatory lesions had thegross appearance of wedge-shaped areas of yellownecrosis and hemorrhage.

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Figure 1. Section of rat kidney 24 hr after inoculation of Proteus mirabilis (H and E, X 196). Thereis selective localization of bacteria in tubular epithelium. The bacterial masses are the darkest stainingmaterial in the section. Note that focal polymorphonuclear reaction about infected tubules is just be-ginning.

Figure JA. Higher power view (X630) of figure 1, showing focal polymorphonuclear reaction beginningabout parasitized tubules.

It was suspected that the unique localizationof Proteus in tubular epithelium was related to ahigh concentration there of urea (the substratefor Proteus urease) and that the necrosis resultedfrom alkalinity secondary to decomposition ofurea by the urease. The series of experiments,which follow, were designed to test these possi-bilities.

Effect of urea concentration on intracellularinfection of renal epithelium by Prote. Tissuecultures of kidney epithelium were better suitedthan the intact kidney for critically examiningthe minute details of intracellular infection byProteus. Confusion between extra- and intra-cellular growth was no problem in tissue culturebecause streptomycin prevented extracellulargrowth. The intracellular growth of Proteus incultures of kidney epithelium 24 hr after inocu-lation is shown in figures 5 and 6. The bacteriaappeared to grow in well organized coloniesconfined to restricted areas of the cytoplasm andpresented the same appearance as the cytoplasmicbacterial colonies found in imprints of the renalepithelial cells of pyelonephritic kidneys (figures2, 2A, and 2B). Their viability was established

by obtaining heavy growth upon bacteriologicalculture of the infected epithelial cells. As shownin table 2, the number of cells infected bycolonies of Proteus increased as the concentrationof urea increased. In other experiments conductedwith higher concentrations of urea, it was foundthat intracellular growth of Proteus was greatestat 0.2 per cent of urea, a concentration approach-ing that in the intact kidney (the average concen-tration of urea in 12 normal rat kidneys was 0.201per cent by weight when determined in kidneyhomogenates of the whole organ). The results intable 2 indicate that concentrations of urea aslow as 0.05 per cent allowed maximal entranceof single bacteria into cells, but that maximalintracellular bacterial growth in the form ofcolonies required higher concentrations of urea.

Unlike Proteus, inoculation of E. coli intotissue cultures produced only slight infection ofmonkey kidney cells. As shown in table 2, only3 to 4 per cent of kidney cells contained coloniesof bacteria and the concentration of urea did notinfluence their growth.

Effect of Proteus urease activity on pH in vivoand in vitro. Because it was postulated that

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selective renal injury from Proteus infection wasrelated to the alkalinity resulting from thedecomposition of the high concentrations of ureapeculiar to that organ, a study was made of the

pH changes that accompanied growth of Proteus,in vivo and in vitro.

(1) Comparison of pH at renal concentrationsof urea with that at extrarenal concentrations

Figure 2. Imprint from rat kidney 12 hr after inoculation of Proteus mirabilis. Intracellular bacterialmultiplication appears to be just beginning in one cell and has developed into a small colony in the other.

Figure 2A. Imprint from rat kidney with lesions shown in figure 1, 24 hr after inoculation of Proteusmirabilis. Note intracellular location of bacterial colonies in cytoplasm of renal epithelial cell (Giemsa,X645).

Figure 2B. Same as 2A except that bacterial colonies are clustered about nucleus of renal epithelialcell.

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Figure S. Section of kidney 24 hr after inoculation of Staphylococcus aureus and (H and E, X140).Bacterial colonies (dark staining masses) are situated between tubules, but never within the tubules asin figure 1. Cellular reaction to S. aureus has not yet started.

Figure 4. Section of kidney 18 hr after inoculation of Escherichia coli (H and E, X 196). Bacterialcolonies (dark staining masses) are situated between the tubules but never within the tubules as infigure 1.

,., .. .. .. ';'' ... .;

.e.::R,.. ~ ~ ~ ~

...-'.'.''..........w;......................._.@. ''................. .if,E......

3@>.........

..... ...%

........~~~~~~~~~~~~~~~ ~~ ~~ ~~ ~~ ~~ ~~ ~~ ~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.

......

Figure 5. Monkey kidney epithelial cell removed from tissue culture 18 hr after inoculation of Proteusmirabilis (Giemsa, X645). Growth of bacterial colony is beginning in cytoplasm of cell. Extracellulargrowth is prevented by streptomycin (50 ,ug per ml).

Figure 6. Same as figure 5 showing growth of several colonies within cytoplasm of cell. Note absenceof extracellular bacteria.

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during growth of Proteus in vitro:-Six strains ofProteus were inoculated into a liquid mediumcontaining serial twofold dilutions of urea inconcentrations ranging from 1.0 to 0.015 percent. The medium was composed of 2 per centpeptone and basic salts. After 18 hr, the pH, asmeasured with a Beckman pH meter, was foundto vary with each concentration of urea, as shownin table 3. The pH values at each concentrationof urea were strikingly uniform for each bacterialstrain and a marked difference was noted betweenrenal and extrarenal concentrations. At 0.015 percent, a concentration found normally in bodyfluids outside the kidney, the pH did not riseabove the normal physiological levels. At thehigh concentrations of 0.125 to 0.25 per centfound in the kidney, the pH rose markedly tovalues ranging from 8.31 to 8.77.

(2) Comparison of urinary pH of Proteuspyelonephritis with that of E. coli pyelonephritis:

TABLE 2Effect of urea concentration on intracellular

infection of monkey kidney cells 24 hr afterinoculation of E8cherichia coli and Proteusmirabilis

Renal Cells* Infected Renal Cells* Infectedwith Bacterial with a Few Dispersed

Conc of Urea in Colonies BacteriaTissue Culture

P. mirabilis E. coli P. mirabilis E. coli

% % ~~% % %0.100 41.0 3.0 21.0 10.00.050 25.0 4.0 36.0 13.00.006 14.0 3.5 9.0 6.0

* Two hundred kidney cells were counted foreach concentration of urea.

-Urines were aspirated with a syringe and needlefrom the bladders of anesthetized pyelonephriticrats 1 week after infection, and the pH of urinesmeasured in a Beckman pH meter with theresults as shown in table 4. The difference in thetwo groups is statistically significant (P = lessthan 0.01).

(3) pH of pyelonephritic lesions during pre-

inflammatory stage:-The pH of pyelonephriticlesions, occurring 18 hr after inoculation of P.mirabilis, was determined by applying the glasselectrode of the Beckman portable pH meter tothe cut surface of the kidney at the site of thelesion and compared with the pH of nondiseasedportions of the same kidney. The condition of thelesions at 18 hr is shown microscopically infigure 1A; it is characterized mainly by selectivebacterial localization in tubular epithelium withtubular necrosis and only minimal inflammatoryreaction. Nine rats were infected and the lesionsin 3 were large enough at 18 hr to provide a

surface that could accommodate the tip of theglass electrode. The following values were noted:Rat No. pH over the Lesion PH of Normal Area

Adjacent to Lesions1 8.1 7.52 8.05 7.63 8.05 7.23

The pH of lesions at 18 hr produced by E. coli(figure 4) were the same as those in normalkidneys (7.3 to 7.7).From these experiments it is concluded that at

the concentration of urea in the kidney, Proteusurease can produce a marked alkalinity that doesnot develop during renal infection by E. coli, andwould not be expected during growth of Proteusin extrarenal tissue.

TABLE 3Comparison of pH at renal and extrarenal concentrations of urea during growth of Proteus in vitro

Conc of Urea (Per Cent)

Proteus Species Renal Extrarenal

1.0 0.5 0.25 0.125 0.0062 0.031 0.015 0.0

P. morganii (Smith). 9.15 8.89 8.77 8.32 7.88 7.47 7.37 7.38P. rettgeri (Stein). 9.18 9.10 8.72 8.31 7.80 7.14 7.28 7.27P. rettgeri (Purbaugh). 9.13 8.38 7.38 7.28P. morganii (Owens) ........... 9.11 8.32 7.45 7.54P. mirabilis (North). 9.12 9.00 8.71 8.32 7.87 7.50 7.33 7.28P. morganii (Biryle). 9.09 8.99 8.72 8.25 77.71 7.43 7.25 7 7.28

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TABLE 4Comparison of urinary pH in Proteus pyelone-

phritis with that in E. coli pyelonephritis

Urine pH ofPyelonephritic Rats

Etiology of Pyelonephritis of RatsRange Mean

Proteus mirabilis ...... 12 7.70-8.52 8.14Escherichia coli ....... 12 6.53-7.90 7.24

Direct injury of renal tissue by urease and by thealkalinity it produces. (1) Production of sterilepyelonephritis by intravascular injection of deadProteus organisms possessing active urease:Suspensions of acetone-killed P. mirabilis, withhigh urease activity, were inoculated intra-cardially in 12 rats and the kidneys massaged inan effort to reproduce the pyelonephritic process

observed with living bacteria. Renal massage hasbeen shown to increase the localization of bacteriain the kidney and does not produce pyelonephritisitself (Braude et al., 1955a). Another group of12 rats was subjected to renal massage afterinoculation with acetone-killed P. mirabilis, butthe urease had been totally inactivated by 0.0008per cent mercuric acetate. Both groups of bacteriahad been washed before inoculation with bufferedsaline to remove acetone and mercury and were

resuspended in trypticase soy broth for inocu-lation. By eliminating urease activity it was

anticipated that the renal injury attributed tothis enzyme could be distinguished from renalinjury by endotoxin and from other enzymes thatare mercury resistant. The catalase in acetone-killed P. mirabilis, for example, was not inacti-vated by the concentration of mercury used toinactivate urease. Mercuric acetate in thisconcentration also failed to reduce the toxicity ofProteus endotoxin extracted by a modifiedBoivin's method (Braude et al., 1955b), from thesame strain of P. mirabilis used in preparation ofthe urease-active bacterial suspensions. Proteusendotoxin, in a concentration of 2 mg per ml, was

exposed to 0.0008 per cent mercuric acetate for24 hr at 28 C and then dialyzed to remove freemercury. The LD5o of the mercury-treated endo-toxin was the same for mice challenged intra-peritoneally as that of the untreated sample ofendotoxin.

One of the 12 rats inoculated with urease-

active suspensions died. All survived in the group

receiving suspensions with inactive urease. Oneweek after inoculation the rats were sacrificedand the kidneys examined. Gross renal lesionswere found in 12 of the 22 kidneys in 11 survivingrats inoculated with bacterial suspensions con-taining active urease. These lesions resembledthose described in rats with acute pyelonephritisdue to living Proteus; they were wedge-shaped,often extended from cortex to pelvis, and onmicroscopic examination consisted of an intenseinterstitial inflammatory reaction with tubularinjury, pus casts, and a tendency to spare theglomeruli.Only 4 of the 24 kidneys in the rats given

mercury-treated suspensions developed grosslesions. The difference in incidence of renal lesionsin the two groups is highly significant (P = lessthan 0.01) as determined by the Chi squaremethod. Failure to eliminate renal injury bytreatment with mercuric acetate may have beendue to reactivation of urease in vivo or to damagefrom mercury-resistant enzymes or endotoxin.To rule out the possibility that the lesions

might be due to infection, one-half of each kidneywas ground to a pulp, suspended in water, andspread across a blood plate for culture at 37 C.All of these cultures proved sterile.

(2) Injury of renal epithelium in tissue cultureby alkalinity created by Proteus urease of viableorganisms:-Growth of P. mirabilis in tissueculture without antibiotics for 24 hr was accom-panied by a gradual increase in pH that wasdirectly related to the concentration of urea.The relationship of urea concentration to pHand renal cell damage is shown in table 5. Kidneycell injury becarme pronounced at 8 hr whenthe pH had remained above 8.0 for 4 hr. At 24hr all cells with concentrations of 0.2 per centurea or more showed injury, but the degree ofdamage to individual cells was much greater in0.3 per cent of urea than in 0.2 per cent. Cellinjury at each concentration was determined byexamining 200 cells on slides stained withGiemsa's stain. At 0.3 per cent the cells had losttheir cell wall, the nuclei had suffered extremepyknosis, and the cells retained the stain poorly.In concentrations of 0.2 per cent at 24 hr, thecell walls were present but disrupted, the nuclearchromatin had lost its fine reticular pattern, thenuclear outlines were irregular and cytoplasmicvacuolization was severe. The cells still stainedshowed fairly good differential staining properties.

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TABLE 5Relationship of urea concentration to pH andkidney cell damage after inoculation of Proteusmirabilis and Escherichia coli in tissue culturesof monkey kidney cells containing varyingconcentrations of urea

Hr afterInfection

0

0

0

0

44448888

24242424

Urea Conc

0.006

0.1000.2000.3000.0060.1000.2000.3000.0060.1000.2000.3000.0060.1000.2000.300

P. mirabilis

pH

7.707.767.747.767.017.678.308.456.237.71 .

8.378.646.478.238.698.89

Damagedkidneycells

2.01.52.02.04.02.51.02.51.09.020.0.17.512.088.0100.0100.0

E. coli

DamagedpH kidney

cellst

7.75 1.07.83 1.07.82 2.07.92 2.06.81 2.56.80 1.06.73 1.06.81 3.06.20 1.06.08 1.06.12 2.56.09 1.06.90 9.06.75 5.06.93 6.06.78 9.0

* Innumerable intracellular and extracellularbacteria present at 24 hr in all concentrations ofurea.

t Innumerable extracellular bacteria present at24 hr in all concentrations of urea.

At concentrations of 0.2 and 0.3 per cent urea

all cells had been torn away from the wall of thetest tube; at 0.1 per cent urea a few normal cellswere still adherent to the glass wall; and at0.006 per cent urea the epithelial layer appearedundisturbed on the wall of the test tube.

Intracellular growth of P. mirabilis in thepresence of extracellular streptomycin (0.005per cent) produced the following changes at 24 hr:

Conc of Urea (%) p Cell Damaged (%)

0.006 7.83 00.10.20.3

7.888.08.20

0

27

These findings demonstrate that intracellulargrowth by itself does not produce apparentinjury unless high alkalinity develops.

Table 5 demonstrates that E. coli, unlikeProteus, produced acidity and almost no celldamage despite heavy growth when inoculated

TABLE 6Injury of kidney epithelium in tissue culture by the

alkalinity produced by urease in the absenceof viable bacteria

Hr after CellsInoculation Type of Urease pH Damagedof Urease

0 None 7.55 1Jack bean, 2 mg 7.67 4Jack bean, 0.2 mg 7.60 5Proteus (acetone-killed 7.00 3suspension)

24 None 7.75 1Jack bean, 2 mg 9.17 70Jack bean, 0.2 mg 8.94 51Proteus (acetone-killed 8.48 64suspension)

into tissue cultures with concentrations of ureaup to 0.3 per cent.

(3) Injury of renal epithelium in tissue cultureby alkalinity created by Proteus urease of deadbacteria, and by Jack bean urease:-The follow-ing experiment was performed to demonstratethat urease alone, in the absence of viablebacteria, could injure kidney epithelium. Jackbean urease, in a concentration of 10 mg per mlwas Seitz-filtered for sterilization and inoculatedinto tissue cultures of monkey kidney epithelium.Another set of tissue cultures of monkey kidneyepithelium were inoculated with 0.5 ml of asuspension of acetone-killed P. mirabilis pos-sessing active urease. The acetone had beenremoved from this suspension by washing withsaline buffered at 7.4. A third set of tissue culturesreceived no urease and served as controls. Alltissue cultures contained 0.2 per cent urea.The results are summarized in table 6. They

show that the alkalinity produced by ureasebreakdown of urea can injure kidney epitheliumseverely in the absence of viable bacteria.

DISCUSSION

These studies suggest that the high concentra-tion of urea in the kidney influences the develop-ment of Proteus infection in 2 ways: (a) byencouraging intracellular infection of tubularepithelium and (b) by undergoing decompositionfrom the action of Proteus urease. The alkalinitythat results is thought to be responsible for theprecipitation of MgNH4PO4 stones and forcellular necrosis.

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The mechanism responsible for attractingProteus to the tubular cells has not been clarified.Staphylococci also possess a potent urease butthese organisms have been observed to developonly outside the tubular cells during the earlystages of pyelonephritis. The conditions re-sponsible for infection of epithelial cells havebeen shown by Shepard (1959) to vary withdifferent bacterial species. It is undoubtedly truethat, while urease and urea concentration in-fluence bacterial infection of renal cells, otherfactors may be equally important.The phenomenon of intracellular infection not

only helps elucidate the initial development ofthe pyelonephritic lesions, but also provides abasis for explaining why Proteus infections persistin the kidney for longer periods than E. coliinfections (Shapiro et al., 1959). The studieswith tissue culture have established that Proteusis protected within the kidney epithelial cellsfrom streptomycin so that bacterial growthoccurs. Their intracellular position could alsoprotect these bacteria from the immune processesthat might otherwise rid them from the tissue.Renal injury might also be expected to progress

after death of Proteus, since the urease activityis preserved in dead bacteria. It is of particularinterest that Proteus killed by streptomycinretains vigorous urease activity. If subsequentstudies bear out the nephrotoxic effect of Proteusurease in pyelonephritis, it would seem that theaction of this enzyme might account not onlyfor the peculiar susceptibility of the kidney tothese otherwise innocuous bacteria, but alsofor the resistance of Proteus pyelonephritis toantibiotics and natural immune processes.

SUMMARY

Evidence has been obtained that Proteusurease is nephrotoxic and that it may contributeto the pathogenesis of pyelonephritis in two ways:(a) by favoring intracellular infection of thetubular epithelium and (b) by creating analkalinity (pH 8.2) in the kidney that leads tonecrosis of renal tubular epithelium and toprecipitation of MgNH4PO4 with formation ofstones.

Intracellular infection was studied in tissuecultures of kidney epithelium containing strep-tomycin to suppress extracellular infection. Intra-cellular infection with Proteus mirabilis increasedas urea concentration rose and was greatest at0.2 per cent, the average concentration in wholekidney homogenates. In the absence of strep-

tomycin, both intra- and extracellular growthoccurred and severe injury to kidney epitheliumappeared with 0.1, 0.2, and 0.3 per cent ureaas the pH rose above 8.2; but no alkalinity andno injury occurred with lower concentrationsof urea. In contrast to Proteus, inoculation ofEscherichia coli produced almost no intracellularinfection in kidney cells either in vivo or intissue culture regardless of urea concentration;no rise in pH in either tissue culture, intactkidney tissue, or urine; and no kidney cell injuryin tissue culture regardless of urea concentration.

Further evidence of Proteus urease nephro-toxicity was obtained by intravascular injectionof acetone-killed P. mirabilis, the urease activityof which was preserved. These urease-activedead Proteus cells produced sterile pyelonephritis.On the basis of these observations, it is suggestedthat urease is one of the important agentsresponsible for the pathogenicity of Proteus inthe kidney.

REFERENCES

BEESON, P. B. AND ROWLEY, D. 1959 The anti-complementary effect of kidney tissue. Itsassociation with ammonia production. J.Exptl. Med., 110, 695-698.

BRAUDE, A. I., SHAPIRO, A. P., AND SIEMIENSKI, J.1955a Hematogenous pyelonephritis in rats.I. Its pathogenesis when produced by a simplenew method. J. Clin. Invest., 34, 1489-1497.

BRAUDE, A. I., CAREY, F. J., SUTHERLAND, D.,AND ZALESKY, M. 1955b Studies with radio-active endotoxin. I. The use of Cr6' to labeledendotoxin of Escherichia coli. J. Clin.Invest., 34, 850.

BRAUDE, A. I., SHAPIRO, A. P., AND SIEMIENSKI, J.1959 Hematogenous pyelonephritis in rats.III. Relationship of bacterial species to thepathogenesis of acute pyelonephritis. J.Bacteriol., 77, 270-280.

RANDALL, A. 1939 The initiating lesions ofrenal calculous. Surg., Gynecol., Obstet.,64, 1-8.

SANFORD, J. P., FAVOUR, C. B., MAO, F. H., ANDHARRISON, J. H. 1956 Evaluation of"positive" urine culture: Approach to differ-entiation of significant bacteria from contami-nants. Am. J. Med., 20, 88-93.

SHAPIRO, A. P., BRAUDE, A. I., AND SIEMIENSKI, J.1959 Hematogenous pyelonephritis in rats.IV. Relationship of bacterial species to thepathogenesis and sequelae of chronic pyelo-nephritis. J. Clin. Invest., 38, 1228-1240.

SHEPARD, C. C. 1959 Nonacid-fast bacteria andHeLa cells: Their uptake and subsequentintracellular growth. J. Bacteriol., 77, 701.

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