![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 284, Issue 3, 1203-1208, March 1998
Section of Urology and Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut
 |
Abstract |
|---|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Systemic bacterial lipopolysaccharides (LPS) induce inflammatory responses characteristic of sepsis. Instillation of LPS into rat bladder produces a localized inflammatory response similar to that seen in urinary tract infections (UTIs). Four hours after intravesical instillation of LPS, neutrophils infiltrate into the bladder, and mRNA for inducible nitric oxide synthase (iNOS) and the cytokines, interleukin (IL)-6 and IL-10, is detected in rat bladder but not in the kidney. Induction of iNOS protein is inferred because urinary nitrate and cGMP levels are increased 4 hr after LPS intravesical instillation and remain elevated for at least 24 hr. When LPS is injected intraperitoneally, iNOS and IL-6 mRNA are induced both in the bladder and in the kidney. These data are consistent with the effects of intravesical instillation of LPS remaining localized. iNOS activity increases in both particulate and soluble bladder fractions when measured 4 hr after intravesical instillation of LPS. The magnitude of these increases in iNOS activity in the bladder is not as great as when LPS is injected intraperitoneally. Intravesical instillation of LPS induces no increase in lung or kidney NOS activity. The localized inflammatory response produced by intravesical instillation of LPS demonstrates the importance of LPS as a mediator of the host response in UTIs and supports the use of urinary measurements of nitrate and cGMP in humans as indicative of the localized induction of iNOS in UTIs.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
During
the course of a human UTI, bacteria ascend into the bladder and attach
to the urothelium, which causes urothelial cells to secrete cytokines
including IL-1
, IL-6 and IL-8 (Svanborg et al., 1994
).
Urinary levels of iNOS (Wheeler et al., 1997), cyclic GMP
(Smith et al., 1996a) and several cytokines, including TNF, IL-1, IL-1
, IL-6 and IL-8 (Hedges et al.,
1992; Ko et al,, 1993; Davidoff et al., 1997)
also are up-regulated during human UTIs, although determining the time
course of induction is difficult. LPS, the cell wall component of many
bacteria including Escherichia coli, can induce many of the
host defenses required for bacterial killing (Cross et al.,
1995) and up-regulates nitric oxide and cytokine production (Szabo and
Thiemermann, 1995) in single cells (Stuehr and Marletta, 1990;
Radomski, et al, 1990), in isolated tissues (Fleming
et al., 1990) and in whole animal models (Knowles
et al., 1990). Intraperitoneal injection of LPS induces the
expression of iNOS protein in many cells and tissues including
mononuclear cells, liver, spleen, lung, kidney and bladder epithelium
(Cook et al., 1994). To determine factors that are necessary
to induce iNOS with conditions that mimic a UTI, we adapted the
procedure of Stein et al. (1996) in which protamine sulfate
followed by E. coli LPS is instilled into rat bladders to
produce inflammation.
We inject LPS intraperitoneally or instill LPS into the female rat
bladder and measure changes in iNOS mRNA and activity in the bladder
and the levels of its enzymatic products including NOx and cyclic GMP
in the urine. Because iNOS is the
Ca++-calmodulin-independent isoform of NOS
whereas the constitutive isoforms nNOS and eNOS are
Ca++-calmodulin dependent, we monitor iNOS
activity as activity in the presence and absence of EGTA. Nitrate and
nitrite are oxidation products of nitric oxide and also are used to
monitor changes in NOS (Evans et al., 1994), whereas cyclic
GMP is increased when NO reacts with the heme moiety of the soluble
guanylyl cyclase enzyme. Cyclic GMP also can be increased
via other pathways including by activation of particulate
guanylyl cyclase enzyme by atrial natruretic factor and related
peptides (Chinkers and Garbers, 1991).
The pro-inflammatory cytokine, IL-6, produces fever and C-reactive
protein response (Nijsten et al., 1987) and up-regulates iNOS in human hepatocytes (Nussler et al., 1995), whereas
IL-10 is an anti-inflammatory cytokine that down-regulates iNOS in rat monocytes (Warner et al., 1995) and macrophages (Cenci
et al., 1993). Thus, we correlated changes in NOS activity
and mRNA with changes in mRNA for cytokines, IL-6 and Il-10, not only
in the bladder and urine, but also in lung and kidney. When LPS is
injected intraperitoneally, systemic changes in NOS and cytokines would be expected not only in the bladder but also in the lung and kidney (Cook et al., 1994), but when LPS is instilled
intravesically, changes in cytokines and iNOS would be expected only in
the bladder.
| |
Methods |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Induction of inflammation. Adult female Sprague Dawley rats (200-250 g) were sedated with xylazine (4 mg/kg) and ketamine (90 mg/kg) and then either injected intraperitoneally with LPS (10 mg/kg) or instilled intravesically with protamine followed by LPS according to our institutionally approved Animal Use protocol. LPS prepared by trichloroacetic acid extraction from E. coli, serotype 026:B6 or serotype 0127:B8, and protamine sulfate, Grade X, was purchased from Sigma, St. Louis, MO.
The procedure of Stein et al. (1996) was followed to instill
LPS into the bladder. After sedation, rats were catheterized with a
lubricated soft catheter (PE20) attached to a syringe. The bladder was
emptied and the urine saved for zero time point measurements. Protamine
sulfate (10 mg) in 500 µl sterile PBS was instilled into the bladder
through the catheter. After 30 min, the bladder was emptied again, and
LPS (1 mg) in 500 µl sterile PBS was instilled through the catheter.
The catheter was removed and the rats allowed to recover. Sterile PBS
(500 µl) or protamine (10 mg in 500 µl sterile PBS) alone was
instilled in control animals. For intraperitoneal LPS injection, 500 µl of 4 mg/ml LPS in sterile PBS was injected. Controls were injected
with 500 µl of sterile PBS. No systemic effects of LPS were noted in
rats in which LPS was instilled.
Urine from control, LPS intraperitoneally injected and LPS
intravesically instilled rats was collected by catheterization or by
gentle manipulation of the abdomen, 4 and 24 hr after LPS treatment,
and animals were sacrificed and their bladders, kidneys and lungs
removed and placed in ice-cold PBS. Bladders from control and treated
rats were fixed in paraformaldehyde and paraffin-embedded sections were
stained with hematoxylin and eosin stain. Leukocytes and epithelial
cells were identified.
Detection of cytokines and iNOS cDNAs after RT-PCR.
Tissues
were homogenized in TRIzol (Life Technologies, Inc, Gaithersburg, MD)
(100 mg tissue/ml TRIzol) and total RNA was extracted. Single-stranded
cDNAs were synthesized with oligo dT priming and Superscript II Reverse
Transcriptase (Gibco BRL, Life Technologies, Gaithersburg, MD).
Species-specific primers for IL-6 (Williams and Coleman, 1995) and
IL-10 (Rabinovitch et al., 1996) allowed amplification of
RT-PCR products at 275 and 436 b.p., respectively. iNOS sense
5
CCCTTCCGAAGTTTCTG GCAGCAG-3 and antisense
5GGGCTCCTCCAAGGTGTTGCCC-3 allowed amplification of a product at
474 b.p. Equivalent amounts of ribosomal protein L19 RT-PCR
product (501 b.p.) (Colin et al., 1995) were amplified in
RNA extracted from bladder and kidney tissue from control rats and from
rats instilled and injected with LPS for 4 hr (data not shown). RT-PCR
products were electrophoresed on 1% agarose gels and stained with
ethidium bromide.
Measurement of urinary NOx levels.
NOx levels were
measured after reduction of nitrate to nitrite with bacterial nitrate
reductase (Schmidt et al., 1992). Nitrate standards (1-100
µM) and rat urines which had been diluted 10-fold in PBS were
incubated (30 min, 37°C) in a reaction mixture (final volume, 100 µl) containing 0.25 U/ml nitrate reductase (Sigma N7265, from
Aspergillus), 250 µM NADPH and 20 µM FAD in PBS. To oxidize NADPH, 10 mM pyruvate and 10 U lactate dehydrogenase (Sigma L2500 from rabbit muscle) were added, the mixture was incubated for an
additional 10 min (37°C) and the nitrite concentration was determined
after addition of 100 µl Greiss reagent. Nitrate was linear over this
concentration range, and there was a 100% conversion of nitrate to
nitrite.
Measurement of urinary cyclic GMP levels.
Cyclic GMP levels
were determined in 1:30 dilutions of urine supernatants by an
125I-radioimmunoassay (Biomedical Technologies,
Stoughton, MA) (Smith et al., 1996a).
Assay of NOS activity.
Tissues were minced and suspended in
10 volumes ice-cold 20 mM HEPES buffer (pH 7.4), containing 1.0 mM
dithiothreitol and protease inhibitors (Dokita et al.,
1994). Bladders were homogenized (10 sec, at 70% power, three times,
and 20 sec at 50% power) by a Polytron (Brinkmann Instruments,
Westbury, NY). Lungs and kidneys were homogenized (10 sec, 70% power)
by an Ultra/Turrax, T25 (IKA Labortechnik, Staufen, KG). All tissue
homogenates were centrifuged (20,000 × g, 20 min,
4°C). The particulate and soluble fractions were stored at 
80°C
until analysis. After thawing on ice, the particulate fractions were
resuspended and washed two times in 10 volumes HEPES buffer plus DTT
and protease inhibitors to remove endogenous L-arginine
(20,000 × g, 20 min, 4°C). The soluble fractions were centrifuged at 50,000 × g for 20 min, and applied
to a 0.5-ml column of AG 50W-X8, Na+ form
(Bio-Rad, Hercules, CA) to remove endogenous L-arginine.
) with bovine 
-globulin as a standard (Bio-Rad, Hercules, CA).
Statistics. Data are presented as mean ± S.E.M. Assessment of LPS effects was obtained by analysis of variance. Significant differences from controls or from one another were obtained with the Scheffé procedure.
| |
Results |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Neutrophil influx. Hematoxylin and eosin-stained sections from bladder from control rats had zero to one leukocyte/high power field. Four hours after intravesical instillation of protamine followed by LPS, hematoxylin and eosin-stained sections of bladder contained approximately 10.7 ± 2.0 leukocytes per high power field (×400, n = 4), and these leukocytes were frequently in or near blood vessels (fig. 1). Some urothelial cell shedding was also visualized at 4 hr. Twenty-four hours after protamine-LPS instillation, urothelial shedding was more prominent, and many leukocytes could be visualized in the subepithelial layer.
|
Changes in iNOS and cytokine mRNA. Four hours after intraperitoneal injection of LPS, iNOS mRNA was detected in the bladder (fig. 2, lane 1) and kidney (fig. 2, lane 4) of female rats. iNOS mRNA was not detected in the bladder (fig. 2, lane 3) and kidney (fig 2, lane 6) of control animals. Intravesical instillation of protamine followed by LPS induced iNOS mRNA in the bladder (fig. 2, lane 2), however, no iNOS RT-PCR product was detected in the kidney (fig 2, lane 5). Intravesical instillation of either LPS or protamine alone did not increase bladder iNOS mRNA.
|
|
Changes in urinary NOx and cyclic GMP levels. Urinary NOx in rats that had been instilled intravesically with LPS after protamine instillation or injected intraperitoneally with LPS increased significantly between 3 and 5 hr, but not at 1 or 2 hr. The level of NOx in the urine of rats before LPS intravesical instillation or intraperitoneal injection was 120 ± 16 nmol/ml. Urinary NOx increased 440% and 580%, respectively, when measured 4 and 24 hr after LPS was instilled intravesically (fig. 4A). Urinary NOx was 84 ± 31 nmol/ml, 24 hr after intravesical instillation with protamine sulfate alone. Four hours after LPS was injected intraperitoneally, urinary NOx increased to 1449 ± 575 nmol/ml.
|
Changes in iNOS activity. In control rat bladder, there was no detectable Ca++-independent particulate or soluble NOS activity. When LPS was instilled into the bladder after instillation of protamine sulfate, Ca++-independent NOS activity was induced in both particulate and soluble bladder preparations. Seventy-two percent of Ca++-independent activity was particulate. When LPS was introduced systemically (injected intraperitoneally), there also was a significant induction in both particulate and soluble Ca++-independent (iNOS) activity in the bladder (fig. 5), 35% of the total activity being particulate.
|
). Small amounts of Ca++-independent NOS
activity (L-NMMA-inhibitable activity) were detected in
control kidney soluble and particulate fractions. Intravesical instillation of LPS after protamine instillation did not significantly change Ca++-independent NOS activity in the
kidney, whereas intraperitoneal injection of LPS increased kidney
Ca++-independent NOS activity 7.3 times in the
soluble fraction (fig. 6A).
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Systemic administration of LPS produces pain, inflammation and
changes in immune function that mimic septic shock. Many of these
effects are mediated by production of cytokines and nitric oxide.
During a UTI, attachment of bacteria to the urothelium causes
epithelial hyperplasia (Uchida et al., 1989), increase in
cytokines (Hedges et al., 1992; Ko et al., 1993;
Svanborg et al., 1994; Davidoff et al., 1997),
influx of neutrophils (Fukushi and Orikasa, 1981) and induction of iNOS
(Wheeler et al., 1997). Intravesical instillation of LPS in
rats after pretreatment with protamine sulfate also causes urothelial
shedding and an influx of neutrophils (Stein et al., 1996).
In our present experiments, we show that LPS induces significant
increases in mRNA message for the cytokines, IL-6 and IL-10, and for
iNOS in the bladder of rats. Bladder iNOS activity, defined as
Ca++-independent NOS activity, also is induced
within 4 hr after intravesical instillation of LPS, with resultant
increases in urinary NO oxidation products and cGMP. Although systemic
(intraperitoneal) LPS treatment up-regulates iNOS and cytokine
production in the bladder and kidney, intravesical LPS treatment does
not induce iNOS or cytokine production in the kidney, which indicates
that intravesical application of LPS remains localized. Neither iNOS
mRNA nor iNOS activity is detected in bladders of control rats.
Systemic injection of LPS produces higher iNOS activity in the bladder
and higher levels of urinary NOx than does intravesical LPS
instillation.
In lung, bladder and kidney, iNOS activity after LPS treatment was
detected in both particulate and soluble fractions. Traditionally, iNOS
was considered a soluble enzyme; however, recent reports (Schmidt
et al., 1992; Wheeler et al., 1997; Moy et
al., 1997) indicate that iNOS is at least partially membrane
associated. iNOS activity in neutrophils isolated from patients with
UTIs is primarily (>90%) particulate (Wheeler et al.,
1997). Transport of eNOS to the caveolae has been found to be important
for function and iNOS has been found to be active only when it is a
dimer (Albakri and Stuehr, 1996). Both post-translational modifications
and localization may affect iNOS activity.
Cytokine production regulates the host response to inflammation and
infection. In bladders instilled with LPS, we have identified mRNA for
both pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines.
Although TNF and IL-1 are regarded as major mediators of septic
shock, IL-1 and to a greater extent TNF production may be transitory
(Ohira et al., 1995). TNF and IL-1, in turn, induce
production of other cytokines such as IL-6. IL-6 levels predict
mortality in septic shock and may represent the net effect of
biologically active TNF and IL-1 (Dinarello, 1996). Davidoff et al. (1997) found that IL-6 is elevated in the urine of
most UTI patients with elevated urinary levels of IL-1. Urinary
cytokine up-regulation has been observed not only in UTI, but also
after intravesical instillation of bacillus Calmette-Guerin for
treatment of superficial bladder cancer, where elevations in urinary
levels of IL-2, IL-6, IL-8 and TNF have been measured (Stassar
et al., 1994). Both epidemiological studies (Kantor et
al., 1984) and animal studies (Davis et al., 1984)
indicate that UTI is a significant risk factor for the development of
bladder cancer and IL-6, which is elevated in UTIs, has been found to
markedly enhance growth of transformed rat bladder cells (Okamoto
et al., 1995).
Anti-inflammatory cytokines like IL-10 are important in counteracting
the effects of LPS and pro-inflammatory cytokines. IL-10 also inhibits
the induction of NOS and improves survival rates in a murine model of
endotoxic shock (Cenci et al., 1993; Thiemermann, 1994;
Warner et al., 1995). IL-10 is up-regulated in the bladder during both intravesical instillation and intraperitoneal injection of
LPS.
Both LPS injection and intravesical instillation induce production of
cytokines and iNOS; however, with intravesical instillation, induction
of both cytokines and iNOS is relegated to the bladder. Previously, we
have measured increases in the urinary levels of iNOS, cGMP, NOx and
IL-8 in patients with UTIs (Smith et al., 1996a; Wheeler
et al., 1997; Saito et al., 1997), kidney
transplant rejection (Smith et al., 1996b) and interstitial
cystitis (Smith et al., 1996a; Wheeler et al.,
1997). From our findings in the rat model, we can infer that changes in
cytokines and NOS-related products during disease processes that affect
the human urinary tract can be measured in the urine of these patients.
In human UTIs, cytokines and iNOS are also induced; however, the time
course of this induction is more difficult to ascertain than with an animal model, where we can observe induction within 4 hr.
Induction of iNOS protein after systemic LPS treatment has been
observed in the bladder urothelium (Cook et al., 1994) where NO production may be involved in bacterial killing (Cross et
al., 1995) and in the regulation of inflammation. Increases in
urinary frequency during UTIs also may be regulated through iNOS,
because in the ileum (Weisbrodt et al., 1996) and in blood
vessels (Schneider et al., 1994), LPS treatment reduces
contractility, and this reduction in contractility is reversed by
treatment with NOS inhibitors. LPS bladder instillation provides a
model to both study and intervene in infectious/inflammatory processes.
| |
Acknowledgments |
|---|
The authors thank Shannon Smith for helpful discussions.
| |
Footnotes |
|---|
Accepted for publication November 14, 1997.
Received for publication August 12, 1997.
1 Supported in part by Public Health Service DK38311 and DK47548 from the National institute of Diabetes and Digestive and Kidney Diseases.
Send reprint requests to: Robert M. Weiss, Section of Urology, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208041, New Haven CT 06520-8041.
| |
Abbreviations |
|---|
b.p., base pair;
EGTA, ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid;
FAD, flavin adenine dinucleotide;
iNOS, inducible nitric oxide
synthase;
HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid;
IL, interleukin;
L-NMMA, NG-monomethyl-L-arginine;
LPS, lipopolysaccharide;
NOS, nitric oxide synthase;
NOx, nitrate plus
nitrite;
PBS, phosphate-buffered saline;
RT-PCR, reverse transcription
polymerase chain reaction;
TNF, tumor necrosis factor;
UTI, urinary
tract infection.
| |
References |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
a-Carde/a G,
Liu J,
Wheeler MA,
Weiss RM and
Sessa WC
(1997)
Subcellular activation and localization of human inducible nitric oxide synthase.
FASEB J
11:
A551.
0022-3565/98/2843-1203$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
This article has been cited by other articles:
|
|
 |
|
  L.-M. Chen, C. Wang, M. Chen, M. R. Marcello, J. Chao, L. Chao, and K. X. Chai Prostasin attenuates inducible nitric oxide synthase expression in lipopolysaccharide-induced urinary bladder inflammation Am J Physiol Renal Physiol, September 1, 2006; 291(3): F567 - F577. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Heemskerk, P. Pickkers, M. P.W.J.M. Bouw, A. Draisma, J. G. van der Hoeven, W. H.M. Peters, P. Smits, F. G.M. Russel, and R. Masereeuw Upregulation of Renal Inducible Nitric Oxide Synthase during Human Endotoxemia and Sepsis Is Associated with Proximal Tubule Injury Clin. J. Am. Soc. Nephrol., July 1, 2006; 1(4): 853 - 862. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-E. Andersson and A. J. Wein Pharmacology of the Lower Urinary Tract: Basis for Current and Future Treatments of Urinary Incontinence Pharmacol. Rev., December 1, 2004; 56(4): 581 - 631. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. S. Kang, F. J. Tamarkin, M. A. Wheeler, and R. M. Weiss Rapid Up-Regulation of Endothelial Nitric-Oxide Synthase in a Mouse Model of Escherichia coli Lipopolysaccharide-Induced Bladder Inflammation J. Pharmacol. Exp. Ther., August 1, 2004; 310(2): 452 - 458. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-E. Andersson and A. Arner Urinary Bladder Contraction and Relaxation: Physiology and Pathophysiology Physiol Rev, July 1, 2004; 84(3): 935 - 986. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. G. Anderson, J. J. Palermo, J. D. Schilling, R. Roth, J. Heuser, and S. J. Hultgren Intracellular Bacterial Biofilm-Like Pods in Urinary Tract Infections Science, July 4, 2003; 301(5629): 105 - 107. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. A. Birder, M. L. Nealen, S. Kiss, W. C. de Groat, M. J. Caterina, E. Wang, G. Apodaca, and A. J. Kanai beta -Adrenoceptor Agonists Stimulate Endothelial Nitric Oxide Synthase in Rat Urinary Bladder Urothelial Cells J. Neurosci., September 15, 2002; 22(18): 8063 - 8070. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. SABAN, H. HELLMICH, N.-B. NGUYEN, J. WINSTON, T. G. HAMMOND, and R. SABAN Time course of LPS-induced gene expression in a mouse model of genitourinary inflammation Physiol Genomics, April 2, 2001; 5(3): 147 - 160. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Franz and W. H. Horl Common errors in diagnosis and management of urinary tract infection. I: Pathophysiology and diagnostic techniques Nephrol. Dial. Transplant., November 1, 1999; 14(11): 2746 - 2753. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Persson, M. Poljakovic, K. Johansson, and B. Larsson Morphological and Biochemical Investigation of Nitric Oxide Synthase and Related Enzymes in the Rat and Pig Urothelium J. Histochem. Cytochem., June 1, 1999; 47(6): 739 - 750. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
