Treatment of Experimental Pyelonephritis in the Monkey

0022-534 7/90/1431-0150$02.00 /0 THE JOURNAL OF UROLOGY

Vol. 143, January

Copyright© 1990 by AMERICAN UROLOGICAL ASSOCIATION, INC.

Printed in U.S.A.

TREATMENT OF EXPERIMENTAL PYELONEPHRITIS IN THE MONKEY JAMES A. ROBERTS,* M. BERNICE KAACK

AND

GARY BASKIN

From The Departments of Urology and Pathology, Tulane University, New Orleans, and the Delta Regional Primate Research Center, Covington, Louisiana

ABSTRACT

Previous studies show that chronic pyelonephritis and end stage renal disease may follow acute pyelonephritis in children and adolescents when improperly or inadequately treated. Our study shows that there is a significant decrease in renal function following untreated acute bacterial pyelonephritis due to nephron loss. The acute inflammatory response is responsible for much of the renal damage, although damage from renal ischemia is an additional significant factor. The present study used a combination of an antibiotic and a xanthine oxidase inhibitor (allopurinol) as compared to antibiotic therapy alone begun 72 hours after infection_ Both were successful in eradicating the infection rapidly, but did not entirely prevent renal damage. Treatment prior to 72 hours thus is important. It appears that the combined treatment, designed to eradicate the bacteria as well as reduce the post-ischemic reperfusion damage and the phagocytic burst of phagocytosis is ideal, as this combined treatment was effective in preventing almost all renal damage and loss of renal function. (J. Ural., 143: 150-154, 1990) Nonhuman primates have been found to have the same incidence of chronic pyelonephritis 1 as that shown in man by Farmer and Heptinstall in comparable autopsy studies. 2 Our experimental studies in the monkey have shown that a single bacterial renal infection leads to chronic pyelonephritis, with a loss of 10 to 20% of the renal mass and function within one month. 3 The loss of function is one which affects glomerular filtration, effective renal plasma flow and proximal tubular function equally (as measured by inulin and P AH clearance as well as Tmax of paraminohippuric acid), confirming the findings of Bricker et al. in the dog who have advanced the "intact nephron theory" as an explanation for nephron loss in chronic pyelonephritis. 4 •5 Stamey has shown that there is, in addition, a distal tubular defect specific for pyelonephritis, wherein the nephrons fail to reabsorb sodium and water. 6 Both Winberg et al. 7 and Smellie et al. 8 have shown the importance of early effective antibiotic therapy in patients with acute pyelonephritis, since they found that both children and adolescents developed renal scarring (chronic pyelonephritis) more frequently when there was a significant delay in the onset of antibacterial therapy of the acute disease. Kunin believes that adults infrequently show renal damage as compared to children. 9 This might be because they have specific symptoms which lead to early effective therapy or their kidneys are not as susceptible to damage from infection as the developing kidneys of children are. The acute inflammatory response is partially responsible for the renal damage. This was shown by Glauser's study in a rodent model wherein he prevented renal damage with early antibiotic therapy. 10 If begun prior to suppuration (no later than 30 hours after infection), no renal scarring occurred. This has been supported by the more recent animal studies of Slotki and Asscher. 11 While they showed that therapy must begin within 24 hours to be protective, Miller and Phillips' similar studies showed that treatment could be delayed for up to four days after the onset of the infection and still be effective in preventing renal scarring. 12 Direct extrapolation of animal data to the clinical situation in man must be done carefully because of interspecies differences in both anatomy and immunological Accepted for publication August 17, 1989 *Requests for reprints: Dept. of Urology, Delta Regional Primate Research Center, Three Rivers Rd., Covington LA 70433. Supported by USPHS grants DK14681 and RR00164.

function. However, it appears that the studies in both animals and man show that early effective antibacterial therapy of acute pyelonephritis is vital to the prevention of chronic pyelonephritis, although exactly when this therapy must be begun to be effective has not been determined. We have studied the mechanisms by which the inflammatory response damages the kidney. Our results showed that renal ischemia leads to reperfusion damage from toxic oxygen radicals 13 as does the respiratory burst of phagocytosis. 14 In both cases superoxide, hydrogen peroxide, hydroxyl radical, singlet oxygen and myeloperoxidase are produced. During phagocytosis these substances are not only released into the phagosome to kill the bacteria but also into the renal tubular lumen where tubular cells are damaged. Following ischemia the oxygen radicals are formed within the tubular cells during reperfusion. We showed the importance of superoxide formation by pre-treatment with both superoxide dismutase 14 and allopurinol 1 3 prior to bacterial challenge. While both treatments led to protection against renal tubular damage from infection, in both instances the protection was not complete even though begun prior to the onset of the infection. Our studies would suggest, however, that amelioration of postischemic damage and that which occurs from the respiratory burst of phagocytosis might improve the results of therapy with antibiotics alone. Thus, the present study is designed to compare therapy with an effective antibiotic, cefonicid, to the combination of cefonicid and allopurinol in the prevention of the renal damage which usually follows untreated acute pyelonephritis in our model.

MATERIALS AND METHODS

Animals. Twenty-one adult female rhesus monkeys (Macaca mulatta) were used in this study. They were fed monkey chow and allowed free access to water. All experimental procedures were done following tranquilization with a mixture of ketamine and xylazine. Voiding cystogram with 20% sodium diatrizoate showed no vesicoureteral reflux. They were then randomly divided into three groups:1) Infection only where eight animals received no treatment; 2) Six animals received 10 days of cefonicid treatment beginning 72 hours after infection; 3) Seven animals received 10 days of

150

AN-TIBIOTIC-Pc,.LLOPURLNOL THERAP-,.1 OF PYELOI\IEPHRJTIS

cefonicid +tion.

Bacterial characteristics. The straic1 of E. coli used in this study (JRl) was isolated from a patient with acute pyelonephritis and has been used in our laboratory. 15 It is serotype 04:Hl, K-non-typeable, has P fimbriae, and type 1 fimbriae when grown in static broth or urine for 16 hours. The bacteria are sensitive to cefonicid, but not killed allopurinol. For the inoculum the bacteria were grown 24 hours on blood agar followed by an 18 hour culture in static broth. The bacteria were centrifuged, washed with sterile saline and diluted to 2x10 9 bacteria/ml. This was combined with 131 I-hippuran and 10% sodium diatrizoate to a final concentration of lxl0 9 bacteria/ ml. inoculum. Method of infection. One kidney was infected by a cystoscopically introduced ureteral catheter, leaving the opposite kidney as a control. The inoculum volume of 0.6 ml. was introduced under fluoroscopic control to assure that pyelotubular backflow occurred. In addition, blood samples taken at one, 10 and 60 minutes were cultured and counted in a gamma counter. These samples showed that pyelovenous inoculation had not occurred. Lipopolysaccharide (LPS) extraction. LPS was extracted from E.coli JRl by the phenol-water extraction method of Westphal and Jann. 16 Fimbriae purification. Fimbriae were purified from E. coli JRl by the method of Korhonen et al. 17 Briefly, bacteria were grown overnight on colonization factor agar. Bacteria were harvested in saline, the fimbriae sheared off in a blender and the whole cells removed by centrifugation at 6,000 g for 30 minutes. Fimbriae were precipitated with 50% ammonium sulfate. The precipitate was collected by centrifugation at 10,000G for one hour and the ammonium sulfate was removed by dialysis against 0.01 M Tris buffer (pH 7.5). Fimbriae were broken into subunits with 0.5% deoxy-cholate. Further purification was on a discontinuous sucrose gradient (60 through 10%) and finally on a Sepharose 4B column equilibrated and eluted with six M urea. Bacteriology and immunology. Urine was obtained suprapubic bladder puncture for culture at zero, two, 24, 48, and 72 hours and at one, two, three and four weeks after infection. Venous blood was taken at these same times for white cell, red cell, and differential white cell counts and serum was stored at -75C for a later determination of anti-P-fimbriae and antibody titers by enzyme-linked immunosorbent assay (ELISA)" Enzyme immunosorbent assay Ninety-six well plates ,~''"'"J"V were coated with 10 mg./ml. of either LPS or fimbriae and incubated in the cold After washing 4X in saline and Tween 20 were overlayed with 5% bovine serum albumin in PBS and incubated one hour at 37C. Serially diluted serum was applied and the incubated three hours at 37C. After washing 4X, anti-human to peroxidase was and the was 45 minutes at room temperature. The was washed 4X with saline-tween before the substrate solution was ~ ~ ~ u , v u at 37C for 15 20 minutes the plates applied. After were read for absorbance on an E.I.A. reader at 490 mn wavelength. Treatment. At 72 hours after infection, treatment was begun in two of the groups as indicated above. Cefonicid was administered intramuscularly at a dose of 0.015 gm./kg./day once a day for 10 days. Allopurinol was administered orally at 200 mg./kg./day once a day for 10 days since that dose had previously been shown to provide some protection against renal damage acutely. 13 Cefonicid was supplied by Smith Kline and French Laboratories, Philadelphia, PA and allopurinol as its sodium base by Burroughs Wellcome Co., Research Triangle Park, NC. Quantitative renal scans. These measures of renal function were done to allow longitudinal studies of the effect of infection without invasive techniques. Standard clearance studies to

151

determine differen.tial renal function would require ureteral catheters which might well complicate the infection. were positioned over the NaI crystal of a General Electric scintillation camera and 50 ,uCi 131 I hippuran given intravenously. An ADAC computer program provided infor-· mation about isotope uptake since renal regions of interest could be indicated and an appropriate area for background subtraction designated at the one to two minute time interval. 18 At that time radionuclide would still be within the renal paren chyma and not in the collecting system. To determine the accuracy and reproducibility of individual renal function measures four monkeys had the studies repeated five times each prior to infection. Sacrifice. Animals were sacrificed by an overdose of barbiturate four weeks after infection. The kidneys were removed with sterile technique and weighed. Half of each kidney was used for culture, the other half being fixed in 10% formalin and stained with hematoxylin-eosin for pathological evaluation. Pathology. The standard section of tissue was taken transversely through the mid portion of the kidney to contain papilla, medulla and cortex. Histological sections were examined by the pathologist in a double-blind manner according to our established histologic parameters. 19 Acute pyelonephritis is associated with a marked inflammatory exudate in areas of bacterial growth, tubular damage and death with microabcess formation. The reparative response with fibrosis and scarring, a mononuclear cell infiltrate especially in the subcapsular, pelvic and periglomerular regions were considered to be subacute to chronic pyelonephritis and are the typical late findings following an untreated infection in our experimental model. Rating was on a 1-4 basis with 4 being the most severe. The sections were rated for the following parameters: tubular and/or interstitial neutrophils, tubular and/or interstitial mononuclears, fibrosis, scarring, pelvic infiltrate, glomerular change, subcapsular invasion, tubular dilatation, and tubular atrophy. In addition the % kidney involved was estimated based on the amount of kidney involved on the H&E slide of the standard section. Statistics. Data among groups was subjected to t test and either one or two way Analysis of Variance for statistical comparison. Multiple comparison was done the NewmanKeuls' Procedure. A one factor repeated measures analysis was used to determine the reproducibility of our measure of renal function. RESULTS

Infection with E.coli led to bacteriuria for 14 ± 9 days however, cleared spontaneously. Treatment with either cefonicid or the cefonicid and allopurinol combination led to resolution of the bacteriuria within three days of the onset of treatment. These values were significantly different from infection alone (ANOVA p=.0027). White blood cell count showed marked elevation within 24 hours of bacterial inoculation, as is usual in our experimental model, with no significant difference in the leukocytosis between treatment groups prior to the initiation of treatment, although there was a significant difference over time for all groups (figure 1). As further evidence for the fact that all monkeys had a renal infection the antibody titer to both P-fimbriae and lipopolysaccharide was shown to increase following infection (table 1). Repeated measurement of % function of each kidney in the four control monkeys who had the study repeated five times over many weeks with our technique of quantitation using radionuclide studies with 131 I-hippuran showed a mean value of 50.0 ± 3.8% per kidney. A one factor repeated measures analysis of these normal kidneys (n=40) showed that the percentage of function over time in control animals did not change (p=0.99). Any change in renal function after infection greater than 7.6% (two standard deviations) would therefore appear valid. The number of monkeys in each group for function studies is less

152

ROBERTS, KAACK AND BASKIN

% RENAL FUNCTION - INFECTED KIDNEY

WHITE BLOOD CELL COUNT

(Polymorphonuclears)

20

.....

Mo

18

o------o

.0

58

6-----6 Inf. + Cefonlcid

-"'z

A---61nf. + Allo.

+ Cefo. (n:;6)

54

Two-way ANOVA: Qroup 1

14

Oh-72h

1w-4w

r::

-

NI

0

p: .0111

O 50

()

.....0

<>-<>infection only (n:6) ~Inf. + Cefonicld (n:6)

- I n f . + Allo. + Cefo.

~ 16 "O 0 0

infection only

C:

::, LL

12

~ 46

:E

C:

0.. 10

0)

'II,

a:

"#. 42 38

0

2h Time After Infection

FIG. 1. Mean white blood cell count/mm. 3 is shown for three groups of monkeys. Note early leukocytosis, which decreases after therapy. There was, however, no significant difference between groups at any time. TABLE

Treatment

Infection Only Infection + Cefonicid Infection + Allopurinol + Cefonicid

Oh

48h

w

w

Time After Infection

4w

1063 83

625 167

11,250 6,486

36,000 9,200

41,500 12,333

45,000 12,167

167

167

8,333

23,333

24,333

7,667

TABLE 2.

Anti-LPS Titers Infection Only Infection + Cefonicid Infection + Allopurinol + Cefonicid

48h

FIG. 2. Mean percentage of total renal function for infected kidneys is shown for three groups of monkeys over time. Normal function for one kidney is 50 ± 3.8% (mean± SD).

l. Mean antibody titers Time After Infection lw 2w 3w Anti-P-Fimbriae Titers

Group one-way ANOVA l:! repeated maaaure11: Infection only p <.0001 Inf. + Cef. pa .0087 30 Inf. + Allo, + Col. NS

63 67

38 33

325 1,383

325 1,600

163 800

188 720

0

0

783

450

450

450

than for pathologic evaluation as some developed bilateral infection as shown by the final pathology. All animals appeared to have had pyelonephritis prior to the initiation of treatment since by 48 hours after infection the function of the infected kidney had dropped in all groups to essentially the same level (fig. 2). The function continued to drop in the monkeys who received either no treatment or antibiotics alone for their infection. However, function began to improve by one week following the infection when the combined treatment regimen (allopurinol plus cefonicid) was given. The infected kidney one month after infection had only 32.3 ± 4 % of total renal function, a significant decrease from normal. Cefonicid alone seemed to cause partial protection as the kidney at one month had 40.2 ± 7% of the total. Allopurinol and cefonicid treatment combined led to a final renal function which was 47.4 ± 6 of the total (fig. 2). Only the combined treatment with allopurinol and cefonicid led to final renal function which was within two standard deviations of normal. A two-way ANOV A showed a significant difference among groups (p <.0001) and also with time (p <.0001). However, only no treatment and cefonicid treatment groups showed decreasing function over time. There

Treatment Infection only Infection + Cefonicid Infection + Allopurinol Cefonicid t-tests of Difference Inf vs Cefonicid Inf vs Allopurinol + Cefonicid

+

Kidney weights (Mean± SD) Infected Kidney

Non -Infected Kidney

df (I-NI)

9.8 ± 1.2 10.8 ± 2.2 10.7 ± 1.3

11.0 ± 1.8 11.7 ± 1.7 10.5 ± 1.3

-1.2 -0.8 +0.3

(df) NS p = .0038

was a significant interaction of groups by time (p=.0041); therefore, a Newman-Keuls' procedure for multiple comparisons was done. This indicated a difference between infection only and infection + cefonicid (p <.05), and a more significant difference between infection only and infection + cefonicid + allopurinol (p <.001). Indeed, comparing infection + cefonicid vs. infection + cefonicid + allopurinol also showed a difference (p <.05). At the time of sacrifice the infected kidney showed chronic pyelonephritis and weighed approximately 10% less than the non-infected kidney. Animals treated with cefonicid showed essentially the same decrease in renal weight due to chronic pyelonephritis and there was no statistically significant difference from that of infection alone. There was no loss of weight of the infected kidneys after treatment with both allopurinol and cefonicid; when compared to the results of infection alone there was a statistically significant difference in renal weights (p=0.0038) (table 2). Pathological evaluation similarly suggested improved results from the combined treatment as compared to cefonicid alone (table 3). While the percentage of kidney involved was only 33% following the combined treatment, 37% was involved in the monkeys treated with cefonicid and 56% was involved when the infection was not treated. Though the combined treatment showed the least involvement,

163 TABL2

3. Pathological evaluation-infected hidney Infection Only (n = 8)

Infection + Cefonicid (n = 6)

Infection+ Allopurinol

+ Cefonicid (n = 7)

ANOVA p=

Neutrophils: Tubular Interstitial Mononuclears: Interstitial Fibrosis Scarring Pelvic Infiltrate Glomerular Involvement Subcapsular Involvement Tubular Dilatation Tubular Atrophy % Involvement

NS

0.63 0.50

0.67 0.08

0.36 0.07

2.50 0.50 2.31 2.75 1.25

2.17 0.50 2.08 1.50 1.17

1.43 0.57 1.71 0.86 0.71

.0137

3.38

2.33

2.00

NS

0.56 2.88 55.9 ±28.3

0.17 2.25 37.6 ±26.l

0.29 1.79 32.6 ±34.1

.0184

NS NS .0006

NS

NS .042 .0882

this was not a statistically significant difference from infection alone. While treatment affected all histologic change, statistically significant protection was shown in four areas scored the pathologist: those being tubular atrophy, pelvic infiltrate, and neutrophillic and mononuclear interstitial infiltrates, all of which were significantly less involved in kidneys from monkeys in the treatment program (table 3). It should be however, that while the combined treatment led to the most protection against pathologic changes, both treatments caused less pathologyo There was a difference between cefonicid alone and the combined treatment Newman-Keuls' procedure for the interstitial mononuclears (p <0005). However, the same test showed a significant difference in tubular atrophy only when comparing infection alone versus the combined treatment with cefonicid and ailopurinol (p <0.05)0 DISCUSSION

VVe chose 72 hours as the time for therapy to commence even though both G!auser's and our previous studies showed the acute inflammatory response had occurred by then. This delay in treatment is longer than the time within which protection was shown by therapy in both Glauser's and Slotki and Asccher's studies but within the time shown by Miller and Phillip's studies. This time was chosen since we feel this may well be the time that the disease becomes clinically diagnosed in man, and would therefore be comparable to therapy of our patients. The antibiotic used was effective in eradicating the infection rapidly but did not totally prevent renal as shown the histologic changes. This is similar to the of the studies of antibiotic treatment in the that suggest that antibiotic treatment is necessary if we are to prevent renal damageo V{hen in our antibiotic treatment was delayed for 72 hours, renal damage was found, thus treatment of pyelonephritis remains of the utmost importance. The addition of allopurinol to the antibiotic therapy cantly decreased some renal pathology and prevented loss of renal weight from tubular atrophy seen after the acute infection, as well as loss of renal function. While the dosage of allopurinol used is high, it was chosen because our previous studies showed that its protective effect was dose dependent and this dosage provided protection against acute damage. 13 In that study we also found that allopurinol had no antibacterial effect, but might affect the respiratory burst of phagocytosis as well as prevent reperfusion damage. The first functional defect from renal infection is a loss of the kidneys's ability to concentrate urine. 20 This defect, however, may be present only temporarily and is found with "acquiesent renal infection," as well as pyelonephritis. 21 Concentrating ability improves as the infection clears as both we and others have shown. 3 •22 It has been shown that permanent renal

functional loss (as deter1nineci filtration may occur in adultso 23 recent shO'vYs that effective renal plasma flow as measured P AH clearance may be reduced even when GFR is normal in children acute pyelonephritis. 24 The abnormality was more common in children developing their infection within the first three years of life. These data confirm the studies of va,1L•Je:,1v. 25 as weil as Michie. 26 Indeed, in children and young adults end stage renal disease the diagnosis of chronic pyelonephritis remains a common one, in incidence for 15 27 to 25%0 28 Those most vulnerable are children with both infection and vesicoureteral reflux. 29 Our studies of high grade reflux in the monkey might as infection caused not only pyelonephritis but also prolonged reflux. 30 Infection has been shown to affect ureteral function other investigators as well.31.a2 In the children and young adults who develop renal failure the loss of functioning nephrons from infection is the basis for the end stage renal disease. In is assumed that as they grow, their renal mass becomes insufficient to maintain normal renal functiono The hypothesis is that increased demand leads to glomerular hyperfiltration of the remaining nephrons with ensuing glomerular sclerosis, an additional insult which ends with renal failure. 33 Brenner's studies that suggest this were done after 5/6 surgical nephrectomy, 33 but we assume that such hyperfiltration may occur after renal damage from infection as welL Our studies enforce the suggestion both Winberg 7 and Smellie 8 that early, effective treatment with antibiotics is all u,,,., n, m, children with py•e!1)rnopJt1nt1s Even might lose renal and oH,w1-,rnolu treated With antibiotiCSo Indeed, in a recent study of treated acute pyelonephritis in adults without urinary tract abnormalities, significant renal damage occurred in over 30% of the patients as determined repeat computed tomodensitomitry scans. 34 While early treatment must be strived for, in our the addition of a drug which decreased both the damage from the respiratory burst of phagocytosis and ischemic damage, allopurinol, was almost all renal damage and prevented effective in loss of renal function. It or some other drug which will decrease the renal ua'"''"" from the toxic oxygen radicals released acute should be considered as a therapeutic adjunct to antibiotic Cefonicid was u~,-,..,H~~. and French Laboratories, its sodium base NC.

cwc~.H>F,U

REFERENCES

L Roberts, J. Clayton, J. D. and Seiboldj I-L R.: rrhe natural incidence of pyelonephritis in the nonhuman prinate. I:D.vest" Urol., 9: 276, 1972. 2. Farmer, E. R. and HeptinstaH, R. H.: Chronic nonobstructive pyelonephritis: a reappraisaL In: Renal Infection and Renal Scarring. Edited by P. Kincaid-Smith Fairley KF. Melbourne, Australia: Mercedes Publishing Pty. Ltd., pp. 233-237, 1971. 3. Janson, K. L. and Roberts, J. A.: Experimental pyelonephritis. V. Functional characteristics of pyelonephritiso Invest Urol., 15: 397, 1978. 4. Bricker, N. S., Dewey, R. R. and Lubowitz, H.: Studies in experimetal pyelonephritis. Simultaneous and serial investigation of a pyelonephritic and intact kidney in the same animal. Clin. Res., 6: 292, 1958. 5. Bricker, N. S., Klahr, S., Lubowitz, H. and Riesselbach, R. E.: Renal function in chronic renal disease. Medicine, 44: 263, 1965. 6. Stamey, T. A. and Pfau, A.: Some functional, pathologic, bacteriologic, and chemotherapeutic characteristics of unilateral pyelonephritis in man. Invest. Urol., 1: 134, 1963. 7. Winberg, J., Andersen, J. H., Bergstrom, T., Jacobsson, B., Larson, H. and Lincoln, K.: Epidemiology of symptomatic urinary tract

154

8. 9. 10. 11. 12. 13. 14.

15. 16.

17. 18. 19. 20.

ROBERTS, KAACK AND BASKIN

infection in childhood. Acta Paediatr. Scand., 252[Suppl] : 1, 1974. Smellie, J. M., Ransley, P. G., Normand, I. C. S., Prescod, N. and Edwards, D.: Development of new renal scars: a collaborative study. Br. Med. J., 290:1957, 1985. Kunin, C. M.: Does kidney infection cause renal failure? Annu. Rev. Med, 36: 165, 1985. Glauser, M. P., Lyons, J. M. and Braude, A. I.: Prevention of chronic pyelonephritis by suppression of acute suppuration. J. Clin. Invest., 61: 403, 1978. Slotki, I. N. and Asscher, A. W.: Prevention of scarring in experimental pyelonephritis in the rat by early antibiotic therapy. Nephron, 30: 262, 1982. Miller, T. and Phillips, S.: Pyelonephritis: The relationship between infection, renal scarring, and antimicrobial therapy. Kidney Int., 19: 654, 1981. Roberts, J. A., Kaack, M. B., Fussell, E. F. and Baskin, G.: Immunology of pyelonephritis VII: Effect of allopurinol. J. Urol., 136: 960, 1986. Roberts, J. A., Roth, J. K., Jr., Domingue, G., Lewis, R. W., Kaack, B. and Baskin, G. B.: Immunology of pyelonephritis in the primate model. V. Effect ofsuperoxide dismutase. J. Urol., 129: 1394, 1982. Roberts, J. A., Kaack, B., Kallenius, G., Miillby, R., Winberg, J. and Svenson, S. B.: Receptors for pyelonephritogenic Escherichia coli in primates. J. Urol., 131: 163, 1984. Westphal, 0. and Jann, K.: Bacterial lipopolysaccharides: extraction with phenol-water and further applications of the procedure. In: Methods in Carbohydrate Chemistry. Edited by R. L. Whistler. New York:Academic Press, pp. 83-91, 1965. Korhonen, T. K., Nurmiaho, E.-L., Ranta, H., Svanborg Eden, C.: New method for isolation of immunologically pure pili from Escherichia coli. Infect. Immun., 27: 569, 1980. Roberts, J. A. and Domingue, G. J.: Experimental pyelonephritis in the monkey. II. The prognostic value of radionuclide evaluation of the urinary tract. Invest. Urol., 12: 374, 1975. Roberts, J. A., Domingue, G. J., Martin, L. N. and Kim, J. C. S.: Immunology of pyelonephritis in the primate model: Live versus heat killed bacteria. Kidney Int., 19: 297, 1981. Winberg, J.: Renal function studies in infants and children with

acute non-obstructive urinary tract infections. Acta Paediatr. Scand., 48: 577, 1959. 21. Miller, T., North, D. and Burnham, S.: Acquiescent renal infection. Kidney Int., 7: 413, 1975. 22. Kaye, D. and Rocha, H.: Urinary concentrating ability in early experimental pyelonephritis. J. Clin. Invest., 49: 1427, 1970. 23. Jacobson, S. J., Lindvall, N. and Lins, L.: Renal size, glomerular function and urinary excretion of albumin and 2-microglobulin in patients with renal scarring due to pyelonephritis. Acta Med. Scand., 222: 261, 1987. 24. Berg, U. B.: Renal dysfunction in recurrent urinary tract infections in childhood. Pediatr. Nephrol., 3: 9, 1989. 25. ·Calcagno, P. L., D'Albora, J.B., Tina, L. U., Papadopoulou, Z. L., Deasy, P. F. and Hollerman, C. E.: Alterations in renal cortical blood flow in infants and children with urinary tract infections. Pediatr. Res., 2: 332, 1968. 26. Michie, A. J., Michie, C. R. and Ragni, M. C.: Kidney function in unilateral pyelonephritis. II. Physiologic interpretations. Am. J. Med., 22: 190, 1957. 27. Scharer, K.: Incidence and causes of chronic renal failure in childhood. In: Dialysis and Renal Transplantation. Edited by J. S. Cameron, D. Fried and C. S. Ogg. Berlin:Pitman Medical Books, pp. 211-217, 1971. 28. The 12th Report of the Human Renal Transplant Registry. JAMA, 233: 787, 1975. 29. Huland, H. and Busch, R.: Chronic pyelonephritis as a cause of end stage renal disease. J. Urol., 127: 642, 1982. 30. Roberts, J. A.: Vesicoureteral reflux in the monkey. A review. Urol. Radio., 5: 211, 1983. 31. Teague, W. and Boyarsky, S.: The effect of coliform bacteria upon ureteral peristalsis. Invest. Urol., 5: 423, 1968. 32. Cox, C. E. II and Elkins, I.: Toxic effects of bacteria on urinary tract smooth muscle. Surg. Forum 19: 524, 1968. 33. Brenner, B. M.: Hemodynamically mediated glomerular injury and the progressive nature of kidney disease. Kidney Int., 23: 647, 1983. 34. Meyrier, A., Condamin, M., Fernet, M., LaBigne-Roussel, A., Simon, P., Callard, P., Ranfray, M., Soilleux, M. and Groc A: Frequency of development of early cortical scarring in acute primary pyelonephritis. Kidney Int., 35: 696, 1989.