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Site of bacterial colonization in acute pyelonephritis induced in rats by Escherichia coli
EBPElII~lENTAL
AND
Site
MOLECULAll
PATHOLOGY
of Bacterial induced TETSUO
Department
Received
253-259 ( 1978)
Colonization in Acute in Rats by Escherichia
SHIMAMIJRA,
JOAN
of Pathology, Rutgers Medical December
29,
COTTON,
College of Medicine School, Piscataway,
20,
1977,
and
Pyelonephritis
co/i1
Jo C. W. WANG
AND
and Dentistry New Jersey
in revised
form
of New 08854
February
23,
Jersey,
1978
An experimental acute pyelonephritis was produced by intravenous or local administration of Escherichia coli, and the kidneys were studied by light and electron microscopy. Obstruction of urinary tract promoted colonization of the bacteria within the kidney. Our data indicate that the colonies of the bacteria were formed initially in the tubular lumina. The bacteria appeared to spread from the tubular lumina into the tubular epithelia and further into the interstitium. Our findings underscore the importance of association between the colonization of the bacteria within the tubular lumina and the obstruction of the urinary tract.
INTRODUCTION In experimental animals, it has been reported that intravenously injected E. cc& became localized in the renal blood vessels (Mallory et al., 1940). These authors concluded that acute pyelonephritis arose from interstitial abscess starting around clumps of bacteria or bacterial thrombi in the small blood vessels. It appears appropriate for us to interpret that these intravascular clumps of bacteria represent the initial site of bacterial colonization in the kidney. Sanford et al., (1962) concluded, however, that the intravenously administered E. coli multiplied within the interstitium, which appears to indicate that the interstitium is the initial site of bacterial colonization. The initial site of bacterial colonization in E. coli induced pyelonephritis does not appear to have been clearly established nor have been extensively investigated. The purpose of our present experiments is to find out the initial site of bacterial colonization within the kidney by E. coli induced acute pyelonephritis in rats. METHODS Nine rats (Holtzman, males) weighing approximately 100 g were anesthetized by inhalation of ether. With thick Nylon thread their left ureters were ligated together with the abdominal muscles (Shimamura et al., 1966). Immediately following the ligation, 0.5 ml Ringer’s solution containing 2 x lOa E. coZi (strain IMRU-54 obtained from the Institute of Microbiology at Rutgers University) per ml was injected through the tail vein. The left ureters of eight control rats were similarly ligated and 0.5 ml of Ringer’s solution without E. coli was in1 This
work
was
supported
in part
by
USPHS
Grant
AM-14411.
0014-4800/78/0292-0253$02.00/O All
Copyright 0 1978 rights of reproduction
by Academic Press, Inc. in any form reserved.
254
SIIIMAMURA,
COTTON,
AND WANG
jetted. Groups of 3, 3, and 3 of the experimental rats and 2, 3, and 3 of the control rats were killed by exsanguination under ether at 24, 35, and 48 hr, respectively, following the injection. A part of the left kidney of each rat was fixed in 1% glutaraldehyde and 4% formaldehyde buffered with monobasic sodium phosphate and sodium hydroxide (pH 7.3) (McDowell and Trump, 1976) for 24 hr, washed overnight with 0.1 M phosphate buffer, post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 1 hr, dehydrated, and embedded in Epon-Araldite mixture No. 1 of Mollenhauer (1964). Th in sections were cut by LKB Ultrotome III, and doubly stained with uranyl acetate and lead citrate for electron microscopy. The remainder of the left kidney and the entire contralateral nonobstructed kidney were fixed in buffered 10% neutral formalin, embedded in paraffin, sectioned, and were stained with hematoxylin eosin and Brown and Brenn’s gram stain for light microscopy. As a separate experiment, left ureters of the three experimental rats were mechanically obstructed as described already, and the left kidney of each rat injected subcapsularly with 2 x 10” of E. coli in 0.2 ml of Ringer’s solution. Two control rats without ureteral obstruction were similarly injected with 2 x 10s of E. coli. Two more rats without ureteral obstruction were similarly injected with 0.2 ml of Ringer’s solution only. All of these rats were killed 24 hr after the injection and the left kidneys were studied by light and electron microscopy as previously described. RESULTS
Hernatogenous
Pyelonephritis
Light microscopic examination revealed that infiltration of polymorphonuclear leukocytes was present in the interstitium and rarely in the tubular lumina at 24 hr. At hours 35 and 48, the infiltration of polymorphonuclear leukocytes became more prominent with a varying degree of destruction of cortical tubular structure. By examination of sections stained with Brown and Brenn’s gram stain, small colonies of gram negative bacilli were observable within occasional tubular lumina at 24 hr (Fig. 1). At 35 and 48 hr, colonies of gram negative bacilli became more prominent in the tubular lumina. In some of the tubules E. coli were spreading into the lining tubular epithelia from the tubular lumina. Besides E. coli in the tubular lumina and in the tubular epithelia, a small number of them were rarely visible in the interstitium after hour 35. Examination of lp-thick sections stained with Paragon Epoxy stain revealed similar topographic distribution of the bacteria as was found in paraffin embedded tissue. In no instance, did the contralateral non-obstructed kidney show any evidence of pyelonephritis. By electron microscopy, the topographic distribution of E. coli was found to be similar to that observed by light microscopy. E. coli were seen within the tubular lumina at hours 24, 35, and 48. After 35 hr, some E. coli were also seen within the interstitium of the cortex. Those observed within the interstitium were usually in the phagolysosomes of the polymorphonuclear leukocytes. In contrast to relatively small number of E. coli found in the interstitium, a large number of E. coZi were seen within the adjoining tubular lumina. It is possible,
EXPERIMENTAL
FIG. 1. Bacterial colonies filtration of polymorphonuclear gram stain. X345.
255
PYELONEPHRITIS
confined within the lumen of a proximal leukocytes is present in the interstitium.
convoluted Brown
tubule. Inand Brenn’s
therefore, to assume that from these tubular lumina which contained a large number of E. cd the bacteria spread into the interstitium through the tubular epithelia and basement membrane. The tubular epithelia, but rarely the tubular basement membrane, were partially destroyed in the area where bacterial spreading occurred from the tubular lumina into the surrounding tissue. Many of the bacteria observed in the tubular lumina revealed close contact with the surface of the brush border (Fig. 2). E. coli were also observed at the base of microvilli of the proximal convoluted tubules (Fig. 2). These E. coli at the base were closely surrounded by brush border microvilli. In addition, there were E. coli that were apparently being engulfed by the tubular epithelia at the luminal surface ( Fig. 2). Furthermore, E. coli were present in the proximal convoluted tubular epithelia within the structures that were compatible with phagolysosomes (Fig. 3). The presence of bacteria in the cytoplasm of proximal convoluted tubular epithelia and in the tubular lumina was more pronounced after hours 35. E. coli contained in the structures that were morphologically compatible with phagolysosomes were much less frequently seen in the epithelia lining distal nephrons. Throughout our experimental time period, we could not detect any colony formation of E. coli within the interstitium; the bacterial colony formation occurred exclusively within the tubular lumina with few exceptions where they were found in the Bowman’s space. At present we are unable to determine how and through what structure these E. coli reach the tubular lumina. Our data, however, indicate that the initial site of formation of bacterial colonies appears to ,be in the tubular lumina from which the bacteria might spread into the tubular epithelia and further into the interstitium. It has been suggested that the proximal tubular epithelia are capable of
256
SHIMAMURA,
COTTON,
AND
WANG
FIG. 2. Twenty-four hours after the injection. Numerous E. coli are visible within the of a proximal convoluted tubule. Many of the E. coli are in close contact with brush microvilli or with luminal cell membrane. Some are in a process of being engulfed tubular epithelia. Microvilli appear to be lost in part. Cytoplasmic vacuoles are seen in the epithelia. X 13,330.
lumen border by the one of
phagocytizing morphologically intact homologous erythrocytes by a process that is apparently independent of pinocytotic protein absorption (Applegate and Tisher, 1973). It is conceivable that the E. coli present within the cytoplasm of the tubular epithelia are probably those that have been phagocytized by the
EXPERIMENTAL
epithelia. Whether or not E. coli are capable epithelia is unknown. Local Injection
257
PYELONEPHRITIS
of actively
invading
the tubular
of E. Coli
Following the subcapsular injection of E. coli, acute pyelonephritis developed only in the kidney whose ureters had been obstructed. We have found that the initial site of formation of colonies of bacteria is within the tubular lumina which is similar to that of the hematogenous pyelonephritis. DISCUSSION One of the important predisposing factors for the development of acute bacterial pyelonephritis is an obstruction of the urinary tract. In our present study, acute pyelonephritis developed in the kidney with ureteral obstruction following hematogenous or local administration of E. coli. Acute pyelonephritis did not develop in the non-obstructed normal kidneys of rats. It seems difficult to infect non-obstructed normal kidneys of rats with such weakly pathogenic organisms as E. coli. Mallory et al. observed initial colonization of hematogenously administered E. coli in the vascular lumina. They also noted an appearance of bacteria in the tubular lumina and in Bowman’s space in slightly older stage of acute pyelonephritis in rabbits, namely, around 48 to 60 hr following the injection of E. coli. In our present study in rats, we could not detect the initial intravascular colonization of E. co& in the pyelonephritic kidneys. Rats are notoriously resistant to infection. The possibility that the rats are capable of destroying intravascular E. coli more efficiently than rabbits could not be ruled out. Localization and fate of E. coli in hematogenous pyelonephritis were reported by Sanford et al. (1962) in rats whose urinary tracts were not obstructed. Their studies included earlier stage of infection than ours. In our present experiment, hematogenous pyelonephritis was produced in rats whose ureters were obstructed. Sanford et al. used fluorescent anti-E. coli antiserum for topographic localization of E. coli within the kidney. It visualized even a small number of E. coli. A possible shortcoming of such a method might be that is could envisage mere presence of E. coEi antigens instead of an aggregate of proliferating E. co& forming a bacteria1 colony. These E. coli antigens could be the products of disintegrated E. coli in the interstitium, or even might be the dilapidated E. coli within the phagolysosomes of leukocytes. Since these leukocytes could emigrate, they could be found at any topographic locations of the kidney. It is difficult to compare our results with those of theirs because of difference in experimental design. The intravenously administered E. coli might initially localize in the vascular system as reported by Mallory et al. ( 1940). The initial location of invasion might well be the vascular walls. It was not clear, however, how the intravenously administered E. coli entered the tubular lumina. In the case of E. coli injected beneath the capsule of the kidney, it was less clear how they found their way into the tubular lumen. E. co& could have reached from the interstitium to the tubular lumina through the tubular epithelia or even through the glomerular capillary waIIs as suggested by Mallory et al. (1940) p ossibly by a means which was similar to emigration of
255
SI~IIhlAMUFiA,
COTTOS,
AND
WANG
Numerous E. coli are present FIG. 3. Thirty-five hours after the injection. epithelia of a proximal convoluted tubule. Several polymorphonuckar leukocytes in the interstitium. They are yet to cross the tubular basement membrane toward luman. No E. coli are seen in the interstitium. X6460.
within the are visible the tubular
leukocytes. Our data not only did not defy such routes of passage of a single or a few E. cali but also were compatible with them. Our emphasis, however, was that these E. coli that entered the tubular lumen might have found it a temporary shelter from the onslaught of leukocytes. In fact at the early stage of the infection, leukocytic infiltration was often virtually absent in the tubular lumina where a mass of E. coli was present. These E. coli were bathed in an ample amount of the intratubular urine due to the presence of hydronephrosis. The urine in these dilated tubular lunrina might have served as good culture media for E. coli (Kass, 1955; Norden and Kass, 1966). Thereby, they were capable of establishing a
EXPERIMENTAL
PYELONEPHRITIS
259
large bacterial colony as early as 24 hr following the injection of the microorganisms. Whereas the E. coli within the capillary lumina or in the interstitium might have been subjected to an immediate onslaught of leukocytes, thereby they failed to establish the initial bacterial colonies in these topographic locations. We have observed E. coli in the tubular lumina without visible bacteria within the adjacent lining tubular epithelia nor in the adjoining interstitium. As much as we could determine, the presence of E. coli within the cytoplasm of the tubular epithelia was always associated with the coexisting intratubular mass of E. coli. These data appeared to indicate that the initial site of colony formation of E. coli was probably in the tubular lumina. In fact, such a phenomenon occurred either by intravenous injection or local administration of E. coli. Recently, evidence was presented which indicated that the brush borders of the proximal convoluted tubules of humans had receptors for E. coli (Scherberich et al., 1977). For E. coli to invade kidneys, attachment of the bacteria to cell membrane might be an essential feature in the development of renal infection. It was not clear, however, whether or not the kidneys of rats had similar receptors for E. coli. Nonetheless, it appeared that E. coli preferentially adhered to the brush borders of the rat kidneys. The mode of spreading of E. coli within the kidney observed in our experiments may be summarized into the following sequences: (1) localization of E. coli in the tubular lumina, (2) replication of E. coli in the tubular lumina, (3) interaction of E. coli with cell membrane of tubular epithelia at the luminal border, (4) phagocytosis of E. coli by the tubular epithelia or “invasion” of the tubular epithelia by E. coli, (5) localization of E. coli within the phagolysosomes of the tubular epithelia, and (6) destruction of the tubular epithelia and tubular basement membrane and spreading of the organisms into the interstitium. Further studies are necessary to elucidate the route and mechanisms by which E. coli find their way into the tubular lumina. REFERENCES APPLEGATE, C. W., and TISHER, C. C. ( 1973). Phagocytosis of morphologically intact erythrocytes by renal proximal tubular epithelium. Amer. J. Putlzol. 70, 2a. KASS, E. H. (1955). Chemotherapeutic and antibiotic drugs in the management of infections of the urinary tract. Amer. J. Med. 18, 764-81. MALLORY, G. K., CRANE, A. R., and EDWARDS, J. E. (1940). Pathology of acute and of healed experimental pyelonephritis. Arch. Pathol. 30, 330-347. MCDOWP.LL, E. M., and TRUMP, B. F. ( 1976). Histologic fixatives suitable for diagnostic light and electron microscopy. Arch. Pnthol. 100, 405414. MOLLENHAUER, H. H. ( 1964). Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111-114. NORDEN, C. W., and KASS, E. H. (1968). Bacteria of pregnancy-a critical appraisal. Ann. Rev. Med. 19, 431470. SANFORD, J. P., HUNTER, B. W., and DONALDSON, P. (1962). Localization and fate of Escherichia cob in hematogenous pyelonephritis. J. Exp. Med. 116, 285-294. SCHERBERICH, J. E., SCHAEFER, K., MONDORF, W., GAUHL, C., and SIETZEN, W. ( 1977). Escherichia coli receptors on human kidney brush-border membranes. Lancet 2, 1181. SHIMAMURA, T., KISSANE, J. M., and GYOERKJIY, F. (1966). Experimental hydronephrosis: nephron dissection and electron microscopy of the kidney following obstruction of the ureter and in recovery from obstruction. Lab. Invest. 15, 629-640.
AND
Site
MOLECULAll
PATHOLOGY
of Bacterial induced TETSUO
Department
Received
253-259 ( 1978)
Colonization in Acute in Rats by Escherichia
SHIMAMIJRA,
JOAN
of Pathology, Rutgers Medical December
29,
COTTON,
College of Medicine School, Piscataway,
20,
1977,
and
Pyelonephritis
co/i1
Jo C. W. WANG
AND
and Dentistry New Jersey
in revised
form
of New 08854
February
23,
Jersey,
1978
An experimental acute pyelonephritis was produced by intravenous or local administration of Escherichia coli, and the kidneys were studied by light and electron microscopy. Obstruction of urinary tract promoted colonization of the bacteria within the kidney. Our data indicate that the colonies of the bacteria were formed initially in the tubular lumina. The bacteria appeared to spread from the tubular lumina into the tubular epithelia and further into the interstitium. Our findings underscore the importance of association between the colonization of the bacteria within the tubular lumina and the obstruction of the urinary tract.
INTRODUCTION In experimental animals, it has been reported that intravenously injected E. cc& became localized in the renal blood vessels (Mallory et al., 1940). These authors concluded that acute pyelonephritis arose from interstitial abscess starting around clumps of bacteria or bacterial thrombi in the small blood vessels. It appears appropriate for us to interpret that these intravascular clumps of bacteria represent the initial site of bacterial colonization in the kidney. Sanford et al., (1962) concluded, however, that the intravenously administered E. coli multiplied within the interstitium, which appears to indicate that the interstitium is the initial site of bacterial colonization. The initial site of bacterial colonization in E. coli induced pyelonephritis does not appear to have been clearly established nor have been extensively investigated. The purpose of our present experiments is to find out the initial site of bacterial colonization within the kidney by E. coli induced acute pyelonephritis in rats. METHODS Nine rats (Holtzman, males) weighing approximately 100 g were anesthetized by inhalation of ether. With thick Nylon thread their left ureters were ligated together with the abdominal muscles (Shimamura et al., 1966). Immediately following the ligation, 0.5 ml Ringer’s solution containing 2 x lOa E. coZi (strain IMRU-54 obtained from the Institute of Microbiology at Rutgers University) per ml was injected through the tail vein. The left ureters of eight control rats were similarly ligated and 0.5 ml of Ringer’s solution without E. coli was in1 This
work
was
supported
in part
by
USPHS
Grant
AM-14411.
0014-4800/78/0292-0253$02.00/O All
Copyright 0 1978 rights of reproduction
by Academic Press, Inc. in any form reserved.
254
SIIIMAMURA,
COTTON,
AND WANG
jetted. Groups of 3, 3, and 3 of the experimental rats and 2, 3, and 3 of the control rats were killed by exsanguination under ether at 24, 35, and 48 hr, respectively, following the injection. A part of the left kidney of each rat was fixed in 1% glutaraldehyde and 4% formaldehyde buffered with monobasic sodium phosphate and sodium hydroxide (pH 7.3) (McDowell and Trump, 1976) for 24 hr, washed overnight with 0.1 M phosphate buffer, post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 1 hr, dehydrated, and embedded in Epon-Araldite mixture No. 1 of Mollenhauer (1964). Th in sections were cut by LKB Ultrotome III, and doubly stained with uranyl acetate and lead citrate for electron microscopy. The remainder of the left kidney and the entire contralateral nonobstructed kidney were fixed in buffered 10% neutral formalin, embedded in paraffin, sectioned, and were stained with hematoxylin eosin and Brown and Brenn’s gram stain for light microscopy. As a separate experiment, left ureters of the three experimental rats were mechanically obstructed as described already, and the left kidney of each rat injected subcapsularly with 2 x 10” of E. coli in 0.2 ml of Ringer’s solution. Two control rats without ureteral obstruction were similarly injected with 2 x 10s of E. coli. Two more rats without ureteral obstruction were similarly injected with 0.2 ml of Ringer’s solution only. All of these rats were killed 24 hr after the injection and the left kidneys were studied by light and electron microscopy as previously described. RESULTS
Hernatogenous
Pyelonephritis
Light microscopic examination revealed that infiltration of polymorphonuclear leukocytes was present in the interstitium and rarely in the tubular lumina at 24 hr. At hours 35 and 48, the infiltration of polymorphonuclear leukocytes became more prominent with a varying degree of destruction of cortical tubular structure. By examination of sections stained with Brown and Brenn’s gram stain, small colonies of gram negative bacilli were observable within occasional tubular lumina at 24 hr (Fig. 1). At 35 and 48 hr, colonies of gram negative bacilli became more prominent in the tubular lumina. In some of the tubules E. coli were spreading into the lining tubular epithelia from the tubular lumina. Besides E. coli in the tubular lumina and in the tubular epithelia, a small number of them were rarely visible in the interstitium after hour 35. Examination of lp-thick sections stained with Paragon Epoxy stain revealed similar topographic distribution of the bacteria as was found in paraffin embedded tissue. In no instance, did the contralateral non-obstructed kidney show any evidence of pyelonephritis. By electron microscopy, the topographic distribution of E. coli was found to be similar to that observed by light microscopy. E. coli were seen within the tubular lumina at hours 24, 35, and 48. After 35 hr, some E. coli were also seen within the interstitium of the cortex. Those observed within the interstitium were usually in the phagolysosomes of the polymorphonuclear leukocytes. In contrast to relatively small number of E. coli found in the interstitium, a large number of E. coZi were seen within the adjoining tubular lumina. It is possible,
EXPERIMENTAL
FIG. 1. Bacterial colonies filtration of polymorphonuclear gram stain. X345.
255
PYELONEPHRITIS
confined within the lumen of a proximal leukocytes is present in the interstitium.
convoluted Brown
tubule. Inand Brenn’s
therefore, to assume that from these tubular lumina which contained a large number of E. cd the bacteria spread into the interstitium through the tubular epithelia and basement membrane. The tubular epithelia, but rarely the tubular basement membrane, were partially destroyed in the area where bacterial spreading occurred from the tubular lumina into the surrounding tissue. Many of the bacteria observed in the tubular lumina revealed close contact with the surface of the brush border (Fig. 2). E. coli were also observed at the base of microvilli of the proximal convoluted tubules (Fig. 2). These E. coli at the base were closely surrounded by brush border microvilli. In addition, there were E. coli that were apparently being engulfed by the tubular epithelia at the luminal surface ( Fig. 2). Furthermore, E. coli were present in the proximal convoluted tubular epithelia within the structures that were compatible with phagolysosomes (Fig. 3). The presence of bacteria in the cytoplasm of proximal convoluted tubular epithelia and in the tubular lumina was more pronounced after hours 35. E. coli contained in the structures that were morphologically compatible with phagolysosomes were much less frequently seen in the epithelia lining distal nephrons. Throughout our experimental time period, we could not detect any colony formation of E. coli within the interstitium; the bacterial colony formation occurred exclusively within the tubular lumina with few exceptions where they were found in the Bowman’s space. At present we are unable to determine how and through what structure these E. coli reach the tubular lumina. Our data, however, indicate that the initial site of formation of bacterial colonies appears to ,be in the tubular lumina from which the bacteria might spread into the tubular epithelia and further into the interstitium. It has been suggested that the proximal tubular epithelia are capable of
256
SHIMAMURA,
COTTON,
AND
WANG
FIG. 2. Twenty-four hours after the injection. Numerous E. coli are visible within the of a proximal convoluted tubule. Many of the E. coli are in close contact with brush microvilli or with luminal cell membrane. Some are in a process of being engulfed tubular epithelia. Microvilli appear to be lost in part. Cytoplasmic vacuoles are seen in the epithelia. X 13,330.
lumen border by the one of
phagocytizing morphologically intact homologous erythrocytes by a process that is apparently independent of pinocytotic protein absorption (Applegate and Tisher, 1973). It is conceivable that the E. coli present within the cytoplasm of the tubular epithelia are probably those that have been phagocytized by the
EXPERIMENTAL
epithelia. Whether or not E. coli are capable epithelia is unknown. Local Injection
257
PYELONEPHRITIS
of actively
invading
the tubular
of E. Coli
Following the subcapsular injection of E. coli, acute pyelonephritis developed only in the kidney whose ureters had been obstructed. We have found that the initial site of formation of colonies of bacteria is within the tubular lumina which is similar to that of the hematogenous pyelonephritis. DISCUSSION One of the important predisposing factors for the development of acute bacterial pyelonephritis is an obstruction of the urinary tract. In our present study, acute pyelonephritis developed in the kidney with ureteral obstruction following hematogenous or local administration of E. coli. Acute pyelonephritis did not develop in the non-obstructed normal kidneys of rats. It seems difficult to infect non-obstructed normal kidneys of rats with such weakly pathogenic organisms as E. coli. Mallory et al. observed initial colonization of hematogenously administered E. coli in the vascular lumina. They also noted an appearance of bacteria in the tubular lumina and in Bowman’s space in slightly older stage of acute pyelonephritis in rabbits, namely, around 48 to 60 hr following the injection of E. coli. In our present study in rats, we could not detect the initial intravascular colonization of E. co& in the pyelonephritic kidneys. Rats are notoriously resistant to infection. The possibility that the rats are capable of destroying intravascular E. coli more efficiently than rabbits could not be ruled out. Localization and fate of E. coli in hematogenous pyelonephritis were reported by Sanford et al. (1962) in rats whose urinary tracts were not obstructed. Their studies included earlier stage of infection than ours. In our present experiment, hematogenous pyelonephritis was produced in rats whose ureters were obstructed. Sanford et al. used fluorescent anti-E. coli antiserum for topographic localization of E. coli within the kidney. It visualized even a small number of E. coli. A possible shortcoming of such a method might be that is could envisage mere presence of E. coEi antigens instead of an aggregate of proliferating E. co& forming a bacteria1 colony. These E. coli antigens could be the products of disintegrated E. coli in the interstitium, or even might be the dilapidated E. coli within the phagolysosomes of leukocytes. Since these leukocytes could emigrate, they could be found at any topographic locations of the kidney. It is difficult to compare our results with those of theirs because of difference in experimental design. The intravenously administered E. coli might initially localize in the vascular system as reported by Mallory et al. ( 1940). The initial location of invasion might well be the vascular walls. It was not clear, however, how the intravenously administered E. coli entered the tubular lumina. In the case of E. coli injected beneath the capsule of the kidney, it was less clear how they found their way into the tubular lumen. E. co& could have reached from the interstitium to the tubular lumina through the tubular epithelia or even through the glomerular capillary waIIs as suggested by Mallory et al. (1940) p ossibly by a means which was similar to emigration of
255
SI~IIhlAMUFiA,
COTTOS,
AND
WANG
Numerous E. coli are present FIG. 3. Thirty-five hours after the injection. epithelia of a proximal convoluted tubule. Several polymorphonuckar leukocytes in the interstitium. They are yet to cross the tubular basement membrane toward luman. No E. coli are seen in the interstitium. X6460.
within the are visible the tubular
leukocytes. Our data not only did not defy such routes of passage of a single or a few E. cali but also were compatible with them. Our emphasis, however, was that these E. coli that entered the tubular lumen might have found it a temporary shelter from the onslaught of leukocytes. In fact at the early stage of the infection, leukocytic infiltration was often virtually absent in the tubular lumina where a mass of E. coli was present. These E. coli were bathed in an ample amount of the intratubular urine due to the presence of hydronephrosis. The urine in these dilated tubular lunrina might have served as good culture media for E. coli (Kass, 1955; Norden and Kass, 1966). Thereby, they were capable of establishing a
EXPERIMENTAL
PYELONEPHRITIS
259
large bacterial colony as early as 24 hr following the injection of the microorganisms. Whereas the E. coli within the capillary lumina or in the interstitium might have been subjected to an immediate onslaught of leukocytes, thereby they failed to establish the initial bacterial colonies in these topographic locations. We have observed E. coli in the tubular lumina without visible bacteria within the adjacent lining tubular epithelia nor in the adjoining interstitium. As much as we could determine, the presence of E. coli within the cytoplasm of the tubular epithelia was always associated with the coexisting intratubular mass of E. coli. These data appeared to indicate that the initial site of colony formation of E. coli was probably in the tubular lumina. In fact, such a phenomenon occurred either by intravenous injection or local administration of E. coli. Recently, evidence was presented which indicated that the brush borders of the proximal convoluted tubules of humans had receptors for E. coli (Scherberich et al., 1977). For E. coli to invade kidneys, attachment of the bacteria to cell membrane might be an essential feature in the development of renal infection. It was not clear, however, whether or not the kidneys of rats had similar receptors for E. coli. Nonetheless, it appeared that E. coli preferentially adhered to the brush borders of the rat kidneys. The mode of spreading of E. coli within the kidney observed in our experiments may be summarized into the following sequences: (1) localization of E. coli in the tubular lumina, (2) replication of E. coli in the tubular lumina, (3) interaction of E. coli with cell membrane of tubular epithelia at the luminal border, (4) phagocytosis of E. coli by the tubular epithelia or “invasion” of the tubular epithelia by E. coli, (5) localization of E. coli within the phagolysosomes of the tubular epithelia, and (6) destruction of the tubular epithelia and tubular basement membrane and spreading of the organisms into the interstitium. Further studies are necessary to elucidate the route and mechanisms by which E. coli find their way into the tubular lumina. REFERENCES APPLEGATE, C. W., and TISHER, C. C. ( 1973). Phagocytosis of morphologically intact erythrocytes by renal proximal tubular epithelium. Amer. J. Putlzol. 70, 2a. KASS, E. H. (1955). Chemotherapeutic and antibiotic drugs in the management of infections of the urinary tract. Amer. J. Med. 18, 764-81. MALLORY, G. K., CRANE, A. R., and EDWARDS, J. E. (1940). Pathology of acute and of healed experimental pyelonephritis. Arch. Pathol. 30, 330-347. MCDOWP.LL, E. M., and TRUMP, B. F. ( 1976). Histologic fixatives suitable for diagnostic light and electron microscopy. Arch. Pnthol. 100, 405414. MOLLENHAUER, H. H. ( 1964). Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111-114. NORDEN, C. W., and KASS, E. H. (1968). Bacteria of pregnancy-a critical appraisal. Ann. Rev. Med. 19, 431470. SANFORD, J. P., HUNTER, B. W., and DONALDSON, P. (1962). Localization and fate of Escherichia cob in hematogenous pyelonephritis. J. Exp. Med. 116, 285-294. SCHERBERICH, J. E., SCHAEFER, K., MONDORF, W., GAUHL, C., and SIETZEN, W. ( 1977). Escherichia coli receptors on human kidney brush-border membranes. Lancet 2, 1181. SHIMAMURA, T., KISSANE, J. M., and GYOERKJIY, F. (1966). Experimental hydronephrosis: nephron dissection and electron microscopy of the kidney following obstruction of the ureter and in recovery from obstruction. Lab. Invest. 15, 629-640.