Full text of DOI:10.1038/labinvest.3780182

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LABORATORY INVESTIGATION
Vol. 80, No. 11, p. 1721, 2000
Copyright © 2000 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
Interstitial Fibrosis of Unilateral Ureteral Obstruction
is Exacerbated in Kidneys of Mice Lacking the Gene
for Inducible Nitric Oxide Synthase
David Hochberg, Christopher W. Johnson, Jie Chen, Drorit Cohen, Joshua Stern,
E. Darracott Vaughan, Jr., Dix Poppas, and Diane Felsen
Center for Pediatric Urology and Laboratory for Minimally Invasive Urologic Surgery, Department of Urology,
Children’s Hospital of New York, Weill Medical College of Cornell University, New York, New York
SUMMARY: Unilateral ureteral obstruction (UUO) is characterized by decreases in renal function and increases in interstitial
fibrosis. Previous studies have indicated that pharmacologic manipulations that increase nitric oxide (NO) are beneficial to the
obstructed kidneys. NO is produced from arginine by nitric oxide synthase (NOS), an enzyme that exists in both constitutive and
inducible (iNOS) forms. To determine the role of the inducible form of NOS in UUO, we used mice with a targeted deletion of iNOS
(iNOS -/- mice) and compared them with wild-type (WT) mice. Kidneys were obstructed for 2 weeks in both WT and iNOS -/- mice,
and were then removed and bisected. Half of the kidney was embedded in paraffin and tissue sections were examined for
interstitial volume or the presence of macrophages. The remainder was flash-frozen and samples were used to measure tissue
collagen (hydroxyproline) or transforming growth factor-␤ (TGF-␤). This study demonstrates that both cortex and medulla of
obstructed kidneys of iNOS -/- mice exhibit significantly increased interstitial volume and interstitial macrophages as compared
with their WT counterparts. Furthermore tissue collagen was increased to 9.2 Ϯ 1.3 ␮g/mg tissue in WT obstructed kidneys,
whereas in iNOS -/- kidneys, collagen was increased to 13.2 Ϯ 0.8 ␮g/mg tissue. The profibrotic cytokine TGF-␤ was also
significantly increased in obstructed kidneys of iNOS -/- mice, as compared with WT mice. No differences were noted between
the unobstructed kidneys of iNOS -/- mice compared with WT mice in any of the parameters examined. These results
demonstrate that targeted deletion of the iNOS results in exacerbation of fibrotic events in the obstructed kidney. These results
confirm previous pharmacologic studies, and suggest that NO produced via the inducible NOS normally serves a protective
function in UUO. (Lab Invest 2000, 80:1721–1728).
enal interstitial fibrosis has been proven to be a
histologic hallmark of chronic ureteral obstruc-
(NOS), which exists in three forms. Neuronal NOS
(nNOS) and endothelial NOS (eNOS) are constitutively
expressed, and there is an inducible isoform (iNOS).
When NOS is induced, iNOS produces large quantities
of NO, which has been implicated in the host response
to sepsis and infection, hemorrhagic shock, and other
disease models (Hierholzer et al, 1998; Kone, 1997).
Because of the abundance of physiologic and
pathophysiologic roles in which NO participates, the
manipulation of NO offers therapeutic potential in a
variety of disease states. Pharmacologic agents that
inhibit NOS can decrease the generation of NO. Se-
lective inhibitors of iNOS commonly used experimen-
R
tion (Gulmi et al, 1998; Klahr and Morrissey, 1998).
Characteristic findings include the expansion of the
renal tubular interstitium with infiltration of monocytes
and macrophages. This is accompanied by the accu-
mulation of both normal and abnormal interstitial ma-
trix proteins, and increases in tissue transforming
growth factor-␤ (TGF-␤) (Diamond et al 1994; Sharma
et al, 1993; Wright et al, 1996). Presumably, fibrosis
develops as an imbalance between interstitial protein
synthesis, deposition, and degradation (Border and
Noble, 1994; Eddy, 1996).
Nitric oxide (NO) has been implicated as a mediator,
messenger, or regulator of cell function in physiologic
situations as diverse as platelet function, vascular
tone, memory, hepatocyte function, apoptosis, and
penile erectile function (Bhagat and Vallance, 1996;
Kim et al, 1999; Kone, 1997). NO is synthesized from
its precursor, L-arginine via the enzyme NO synthase
tally
include
aminoguanidine
and
L-N6-(1-
iminoethyl)lysine (Bryk and Wolff, 1998; Moore et al,
1994). Newer mechanism-based inactivators of NOS,
which require catalytic turnover to irreversibly inacti-
vate NOS, have been described; in addition, allosteric
inhibitors of NOS have been identified by combinato-
rial chemistry (Bryk and Wolff, 1999; McMillan et al,
2000). At issue with the use of NOS inhibitors in vivo is
not only the cross-reactivity between the various
forms of NOS, but also the pharmacologic side effects
of these agents.
It has been recently recognized that increased NO
generation is a common characteristic of certain phar-
macologic agents that ameliorate interstitial fibrosis.
Received July 12, 2000.
Address reprint requests to: Dr. Diane Felsen, Associate Research Professor of
Pharmacology in Urology, Weill Medical College of Cornell University,
Department of Urology, Box 94, 1300 York Avenue, New York, New York
1
0021. Fax: 212 746 3314; E-mail: dfelsen@med.cornell.edu
Laboratory Investigation • November 2000 • Volume 80 • Number 11 1721

Hochberg et al
The administration of arginine and angiotensin con-
verting enzyme (ACE) inhibitors have both been found
to blunt the fibrotic response to unilateral ureteral
obstruction (UUO) (Ishidoya et al, 1995; Morrissey et
al, 1996). Arginine is a direct precursor to NO, whereas
ACE inhibitors are thought to augment NO production
indirectly by stimulating bradykinin production (Zhang
et al, 2000). Comparable evidence also exists in that
the pharmacologic inhibition of NOS during UUO
exacerbates the resulting interstitial fibrosis (Morrissey
et al, 1996). Thus NO appears to be protective, but the
molecular source of the NO is unknown. To avoid
some of the difficulties associated with pharmacologic
inhibition of NO, and to more specifically evaluate the
role of iNOS in the fibrosis that results from UUO, we
have used mice with a targeted deletion of the iNOS
gene (iNOS -/- mice) to study interstitial fibrosis in
UUO.
-/- mice. In Figure 1 are shown trichrome stained
sections from these kidneys. In Figure 1, a and b, are
shown the unobstructed kidneys of WT (Fig. 1a) and
iNOS -/- mice (Fig. 1b). Kidneys from both types of
mice show a regular arrangement of tubules with little
interstitial fibrosis. There is little apparent difference
between kidneys from WT and iNOS -/- mice. UUO is
accompanied by tubular dilatation, associated with a
marked increase in interstitial collagen deposition
(blue staining) in both WT kidneys (Fig. 1c) and iNOS
-/- kidneys (Fig. 1d) (compare Fig. 1a with b, and c
with d). The kidneys of obstructed iNOS -/- mice
exhibited increased interstitial volume, interstitial fi-
brosis, and interstitial cellularity as compared with WT
mice (compare Fig. 1c with d), as will be detailed and
quantified below.
Interstitial Volume
Results
Interstitial volume expansion in response to UUO has
previously been shown in both rat and rabbit kidney
Histologic Analysis
(Sharma et al, 1993; Wright et al, 1996). In the present
In the present study we compared the response to
UUO in both control unobstructed kidneys and in
obstructed kidney from both wild-type (WT) and iNOS
study we confirm that a similar change occurs in the
mouse kidney. In WT mice there was a significant
increase (2.5 times compared with control) in cortical
Figure 1.
Masson’s trichrome staining of mouse kidney sections. Paraffin sections, 5-␮m thick, were stained with Masson’s trichrome. The blue-appearing collagen is
particularly evident in obstructed specimens. a, Control wild-type(WT) kidney; b, control inducible nitric oxide synthase (iNOS) -/- kidney; c, 14-day obstructed WT
kidney; d, 14-day obstructed iNOS -/- kidney. Original magnification, ϫ400.
1
722 Laboratory Investigation • November 2000 • Volume 80 • Number 11

Interstitial Fibrosis of UUO in iNOS -/- Mice
interstitial volume at 14 days of obstruction (Table 1).
Medullary interstitial volume was also significantly
increased.
We found that the interstitial volume expansion was
significantly greater (p Ͻ 0.05) in both the cortex and
medulla of obstructed iNOS -/- kidneys compared
with the obstructed WT (Table 1). After obstruction,
cortical interstitial volume increased 3.2-fold in kid-
neys of iNOS -/- mice as compared with a 2.5-fold
increase in kidneys of WT mice.
No significant difference, quantitative or qualitative,
was appreciated between control WT and iNOS -/-
kidneys. Control WT cortical interstitial volume (%)
was 11.4 Ϯ 1.9, whereas control iNOS -/- interstitial
volume was 11.1 Ϯ 0.8 (Table 1). No differences were
detected in the medulla (Table 1).
obstruction (Table 3). The number of F4/80-positive
cells per high power field increased by 20.9 and 26.0
cells in the cortex and medulla of the WT kidney,
respectively. Increases in iNOS -/- cortical and med-
ullary F4/80-positive cells were 34.2 and 41.4 cells per
high power field, respectively.
Furthermore the number of F4/80-positive cells was
found to be significantly greater in both cortex and
medulla of obstructed kidneys of iNOS -/- mice com-
pared with WT (Table 3). Comparison of control WT
with iNOS -/- kidneys illustrated no significant
differences.
Discussion
Interstitial fibrosis includes the accumulation of inter-
stitial matrix proteins, inflammatory cell infiltrates, and
the production of proinflammatory cytokines that
leads to a complex and dynamic imbalance among
interstitial matrix synthesis, deposition, and degrada-
tion. Interstitial fibrosis is the histologic hallmark of
virtually all progressive renal diseases, including
chronic ureteral obstruction, and it is the single best
histologic correlate of decline in renal function and
long-term prognosis (Eddy, 1996). Although previous
studies have implicated a role for NO in ameliorating
fibrosis during UUO, the enzymatic source of NO has
not been determined. The present study used iNOS -/-
mice to probe the role of inducible NOS in this
process. Our results demonstrate that interstitial fibro-
sis is exacerbated in both the cortex and medulla of
iNOS -/- mice after 14 days UUO compared with WT
mice. UUO in iNOS -/- mice is accompanied both by
significantly greater increases in interstitial volume,
tissue collagen, and TGF-␤ and by interstitial macro-
phage infiltration. This suggests that the NO synthe-
sized by the inducible form of NOS, may normally be
protective in UUO.
Previous investigators have suggested roles for NO
in models of fibrosis in other systems. By using
L-NAME in the drinking water of rats, the chronic
inhibition of NOS has been shown to increase angio-
tensin II-induced cardiac fibrosis (Hou et al, 1995).
Furthermore the chronic treatment of rats with
L-NAME has been shown to result in both myocardial
and perivascular fibrosis (Numaguchi et al, 1995). In
contrast other authors have been unable to confirm
these findings using similar models (Sigusch et al,
1996).
Tissue Collagen
Tissue collagen, as measured by analyzing tissue
hydroxyproline (see “Methods”) was also assessed in
kidneys of WT and iNOS-/- mice. At 14 days there
were significant differences in the collagen content of
the obstructed WT and iNOS -/- kidneys as compared
with their respective control kidneys (Table 2). Further-
more the obstructed iNOS -/- kidney contained more
collagen than the obstructed WT kidney (13.1 Ϯ 0.8
compared with 9.2 Ϯ 1.3 ␮g/mg tissue dry weight;
Table 2).
Tissue TGF-␤
Since TGF-␤ has been considered to be a profibrotic
cytokine with a role in UUO, we examined tissue
TGF-␤ using the mink lung bioassay. In WT mice at 14
days, tissue TGF-␤ was increased by 6.8-fold in the
obstructed kidney relative to its control(Table 2). In
comparison, there was more than a 28-fold increase in
tissue TGF-␤ with obstruction in iNOS -/- mice. Tissue
TGF-␤ in the obstructed kidney of iNOS -/- mice was
significantly greater than that in obstructed kidney of
WT mice.
Macrophage Quantification
An increase in interstitial cellularity in particular mono-
cytes and macrophages is one of the primary features
of interstitial fibrosis. To identify macrophages we
used a monoclonal antibody to the F4/80 antigen, a
murine macrophage-specific cellular membrane
marker that maintains its antigenicity during parafor-
maldehyde fixation and paraffin embedding (Gordon
et al, 1992).
In the kidney the role of NO in models of ureteral
obstruction and other diseases is complex. The in-
volvement of NO has often been studied using argi-
nine supplementation. Use of arginine administration
(either through dietary supplement or by injection) has
WT and iNOS -/- mice both showed significant
increases in F4/80-positive cells after 14 days of
Table 1. Interstitial Volume (%) in iNOS ؊/؊ and Wild-Type (WT) Mice at 14 Days of UUO
WT control
WT-obstructed
iNOS Ϫ/Ϫ control
iNOS Ϫ/Ϫ obstructed
Cortex
Medulla
11.4 Ϯ 1.9
15.8 Ϯ 2.3
28.7 Ϯ 3.3*
32.7 Ϯ 1.5*
11.1 Ϯ 0.8
18.5 Ϯ 1.2
35.4 Ϯ 2.1*‡
43.7 Ϯ 1.5*‡
*
p Ͻ 0.05 compared to unobstructed control; ‡ p Ͻ 0.05 compared with WT obstructed.
Laboratory Investigation • November 2000 • Volume 80 • Number 11 1723

Hochberg et al
a
Table 2. Tissue Collagen and Tissue TGF-␤ in iNOS ؊/؊ and WT Mice
WT control
WT-obstructed
iNOS Ϫ/Ϫ control
iNOS Ϫ/Ϫ obstructed
Collagen
TGF-␤
4.6 Ϯ 0.5
2.4 Ϯ 0.3
9.2 Ϯ 1.3*
16.5 Ϯ 1.0*
7.8 Ϯ 0.7
1.6 Ϯ 0.9
13.2 Ϯ 0.8*‡
45.0 Ϯ 3.6*‡
*
p Ͻ 0.05 compared to unobstructed control; ‡ p Ͻ 0.05 compared with WT obstructed.
Collagen, ␮g/mg tissue, dry weight; TGF-␤, pg/mg tissue, wet weight.
a
Table 3. F4/80-Positive Cells per High Power Field
WT control
WT-obstructed
iNOS Ϫ/Ϫ control
iNOS Ϫ/Ϫ obstructed
Cortex
Medulla
0.5 Ϯ 0.2
1.2 Ϯ 1.0
21.4 Ϯ 0.9*
27.2 Ϯ 6.0*
1.7 Ϯ 0.9
2.9 Ϯ 0.3
35.9 Ϯ 1.3*‡
44.3 Ϯ 1.7*‡
*
p Ͻ 0.05 compared with unobstructed control; ‡ p Ͻ 0.05 compared with WT obstructed.
resulted in amelioration of fibrosis in models of sub-
total nephrectomy, diabetes, hypertensive nephro-
sclerosis, and cyclosporine nephrotoxicity (Chen et al,
ministration has been shown to enhance recovery of
renal function (Reyes et al, 1992). The present study is
in agreement with previous reports in that we demon-
strate an exacerbation of fibrosis when the gene for
iNOS is deleted. The general protective role of NO in
UUO is also supported by other studies from this
laboratory in which the apoptosis of UUO is signifi-
cantly exaggerated in iNOS -/- mice (Miyajima et al,
1993; De Nicola et al, 1993; Katoh et al, 1994; Reyes
et al, 1993). In glomerulonephritis, arginine supple-
mentation has both ameliorated and exacerbated the
disease (Peters et al, 1999, 2000). The timing of
administration of arginine may be a critical factor in its
effects. Beneficial effects of the administered arginine
may result from its metabolism to NO. The resulting
NO may by itself be antifibrotic (Trachtman et al,
2000b).
In contrast, the present results, and those cited
above using pharmacologic manipulation of NO, are in
conflict with those recently reported by Huang et al
1996). In addition metabolism of arginine to NO by
endothelial NOS may result in reversal of vasocon-
strictive effects caused by previously decreased argi-
nine. In contrast detrimental effects of arginine admin-
istration may be caused by synthesis of large
quantities of NO or other toxic metabolites resulting
from metabolism of arginine by iNOS, which could be
responsible for tissue damage. In addition the possi-
bility that arginine supplementation results in metab-
olism of arginine to non-NO products also needs to be
considered in assessing both beneficial and harmful
effects of arginine supplementation.
In UUO specifically, several studies have demon-
strated a protective role for NO. To pharmacologically
increase the production of NO, rats were treated with
ACE inhibitors, or a dietary supplement of L-arginine.
Both of these agents were confirmed to increase NO
generation, as demonstrated by increases in urinary
nitrite concentration. It was shown that experimentally
obstructed rats given each of these agents displayed
significantly less interstitial fibrosis after UUO. Further-
more the pharmacologic inhibition of NOS with
L-NAME not only reversed the beneficial effects when
used with the aforementioned agents, but when used
alone, the interstitial fibrosis of UUO was significantly
exacerbated (Morrissey et al, 1996). Low levels of
arginine have been found associated with bilateral
ureteral obstruction (Reyes et al, 1994); in studies on
the effects of arginine on renal blood flow in a canine
model of UUO, we found that decreased renal function
in UUO may be related to a decreased tissue supply of
arginine (Schulsinger et al, 1997). Furthermore, in a
study examining the unilateral release of obstruction in
a model of bilateral ureteral obstruction, arginine ad-
(
2000). Using iNOS -/- mice with UUO, they reported
decreased fibrosis as assessed by trichrome staining
and collagen I deposition. In addition, the cortex of
kidneys of iNOS -/- mice with UUO was thinner than
the cortex of WT mice with UUO, suggesting that
cortical thinning is associated with decreased fibrosis.
However, using MRI in patients it has been shown that
the renal cortex of patients with interstitial fibrosis is
thinner than cortex of normal controls (Kettritz et al,
1
996), which is contradictory to the finding of Huang et
al. It is unclear at present why there are discrepancies
between these studies.
The majority of the studies of NO in models of
fibrosis suggest that NO is antifibrotic, and conversely
that inhibition of NO is profibrotic (Hou et al, 1995;
Morrissey et al, 1996; Tomita et al, 1998). Because of
the lack of specificity of the pharmacologic NOS
inhibitors used in these studies, a clear determination
of the impact of each specific NO synthase in UUO
has yet to be illustrated. Thus the present study, using
iNOS -/- mice, not only confirms that a lack of NO is
associated with exacerbation of fibrosis, but it also
serves to implicate the inducible form of NOS in this
process. Studies using iNOS -/- mice are thus able to
establish a role for the inducible NOS, which can only
be suggested using pharmacologic studies. This has
been shown in several different disease models
(Mashimo and Goyal, 1999).
Furthermore, unlike previous studies using iNOS
inhibitors (Morrissey et al, 1996), we do not appreciate
any significant differences between the iNOS -/- and
WT control kidneys. Neither histologic appearance nor
1
724 Laboratory Investigation • November 2000 • Volume 80 • Number 11

Interstitial Fibrosis of UUO in iNOS -/- Mice
tissue collagen or interstitial volume are different in
iNOS -/- as compared with WT mice. This suggests
that previous differences found by using pharmaco-
logic inhibition of NOS may be attributed to the effects
of these agents on the constitutively expressed NO
synthases, or to other nonspecific effects of these
drugs.
Results of tissue TGF-␤ analysis confirm that the
obstructed kidney contained increased TGF-␤ as
compared with the control kidney, as has been previ-
ously shown in rats (Wright et al, 1996). Furthermore,
the TGF-␤ is further increased in iNOS -/- mice. This
finding is in agreement with previous studies using
L-NAME treatment of rats in a cardiac fibrosis model.
In that model, treatment with L-NAME leads to in-
creased expression of TGF-␤ and extracellular matrix
proteins after L-NAME administration (Tomita et al,
Materials and Methods
iNOS -/- Mice
Mice with a targeted deletion of the iNOS gene were
obtained from Dr. Carl Nathan, Cornell University
Medical College. The iNOS gene was inactivated
through homologous recombination using a replace-
ment vector targeted to delete the proximal 585 bases
of the iNOS promoter. The extent and selectivity of
iNOS deficiency in vivo were confirmed by Northern
and Southern blots. Southern blots demonstrated a
single iNOS allele in WT animals, which was not
detected in iNOS -/- mice. iNOS was unable to be
induced, whereas eNOS and nNOS expression was
neither diminished nor increased in compensation for
iNOS deficiency (MacMicking et al, 1995).
1
998). Another striking finding is the significantly
greater increase in macrophage infiltration in the iNOS
/- mice. In vivo, NO has been shown to inhibit
Tail Clip DNA and PCR
-
In the present study, the NOS genotype was deter-
mined by PCR using tail clip DNA. Tails were cut into
0.4–0.6-cm pieces and DNA isolated using a QIAamp
kit from Qiagen (Valencia, California). Briefly, samples
were incubated in Proteinase K, centrifuged, and
incubated in buffer containing ethanol. RNAse was
added to the sample, which was then spun on a
QIAamp spin column.
PCR was carried out as described (MacMicking et
al, 1995) for 35 cycles with Taq polymerase (0.75 U) at
an annealing temperature of 60° C (30 seconds) with
primer extension at 72° C (3.0 minutes). Seventy
nanograms of DNA was used per reaction, and prim-
ers were specific for either WT iNOS (PCR product,
leukocyte adhesion to the endothelium of blood ves-
sels (Kanwar and Kubes, 1995). Conversely, the inhi-
bition of NOS has been shown to elicit significant
leukocyte adhesion (Akimitsu et al, 1995). In vitro,
inhibition of NO with pharmacologic NOS inhibitors
up-regulates the mRNA of the monocyte chemoat-
tractant protein, MCP-1, which plays a role in mono-
cyte recruitment (Zeiher et al, 1995). Recent studies
using iNOS -/- mice have demonstrated similar find-
ings in two different colitis models. In both the TNB
and DSS models of colitis, iNOS -/- show increased
severity and greater damage as compared with WT
animals. These results support a role for iNOS in
protecting against inflammation (Beck et al, 1999; Qiu
et al, 1998).
The mechanism by which interstitial fibrosis causes
compromise of renal function is not clearly under-
stood. A significant factor is likely to be the ultimate
obliteration of the renal tubules by the fibrogenic
process (Eddy, 1996). Also, destruction of postglo-
merular interstitial capillaries, as well as the overex-
pression of vasoconstrictors, is likely to compromise
the oxygen supply to the interstitium, thus worsening
renal function (Eddy, 1996). This may help to explain
the importance of NO, especially its vasodilatory ef-
fects, during chronic ureteral obstruction.
In summary, our data demonstrate that interstitial
fibrosis is exacerbated in the obstructed kidneys of
mice lacking the gene for inducible NO synthase.
iNOS -/- mice showed evidence of significantly greater
interstitial fibrosis after UUO, as assessed by in-
creases in interstitial volume, collagen, TGF-␤, and
macrophage infiltration. These data therefore suggest
that, among its myriad of pathophysiologic functions,
inducible NO serves a protective role during chronic
UUO. With the advent of newer, more specific phar-
macologic manipulation of NO, strategies that aug-
ment the generation of nitric oxide may soon prove
beneficial as antifibrotic agents during chronic ureteral
obstruction.
413 bp) or the iNOS -/- (PCR product, 1288 bp), which
was visualized as a mutant form. The PCR products
were then separated on an agarose gel and products
were visualized with ethidium bromide. In Figure 2 is
shown a composite PCR that was run using tail clip
DNA from both iNOS -/- and WT mice. Lanes 1–3,
from iNOS -/- mice, show a single band that corre-
sponds to the standard for iNOS -/-, shown in Lane 4
and Lane 13. DNA from WT mice is shown in Lanes
5–7; the PCR product is of a different size and
corresponds to that found with a cDNA standard (data
not shown). The samples in Lanes 8–12 are derived
from tail clip DNA from WT mice probed with primer
for the iNOS -/- to demonstrate that WT animals do
not exhibit the iNOS -/- genotype.
Ureteral Obstruction
WT and iNOS -/- mice (n ϭ 6 per group) underwent
right unilateral ureteral ligation with 4–0 silk suture
through an abdominal midline incision under sterile
conditions. Animals were anesthetized with 0.12–0.15
cc intraperitoneal pentobarbital (6.5 mg/ml). Sham-
operated animals underwent identical operations,
however with manipulation of the right ureter only.
Animals were housed in groups of 3 to 5 and fed
standard chow and water ad libitum. At 14 days, both
obstructed and control kidneys were removed follow-
Laboratory Investigation • November 2000 • Volume 80 • Number 11 1725

Hochberg et al
Figure 2.
Confirmation of genotype in WT and iNOS -/- mice. Reactions were run using 70 ng of tail clip DNA as described in “Materials and Methods.” The PCR product for
WT is 413 bp; the PCR product of the iNOS -/- is 1288 bp. Samples: Lanes 1 to 3, tail clip DNA from three separate iNOS -/- mice; Lane 4, cDNA std, iNOS -/-; Lanes
5
to 7, tail clip DNA from three separate WT mice probed with WT primers; Lanes 8 to 10, tail clip DNA from WT mice probed with iNOS -/- primers; Lane 13, cDNA
std, iNOS -/-; Lane 14, PCR blank, no DNA; Lane 15, mw markers.
ing the administration of IP pentobarbital. Animal
treatment adhered to approved institutional
guidelines.
TGF-␤ Measurements
Mink lung epithelial cells (MLECs), which were trans-
fected with a PAI-1 promoter driving expression of
luciferase in a TGF-␤ responsive manner, were used
for analysis of TGF-␤ 1 activity as previously described
1
Renal Morphology
(
Miyajima et al, 2000a). These cells were a generous
Sections of hemisected obstructed and contralateral
kidneys were fixed in 10% neutral buffered formalin
and embedded in paraffin. Sections 5-␮m thick were
stained with hematoxylin and eosin and Masson’s
trichrome for light microscopic analysis. Histologic
studies were performed in a blinded fashion, with
treatment group unknown to the examiner.
gift from Dr. Daniel Rifkin. Transfected MLEC cells
were plated in 96-multiwell plate at 37° C in a humid-
ified incubator. The TGF-␤1 in the test sample was
activated with 1N HCl (1 hour at 4° C), followed by
equilibration with 1N NaOH. After 3 hours, the medium
was replaced with the activated test sample and
incubated overnight at 37° C. Cells were then lysed
and luciferase reagent (Promega, Madison, Wiscon-
sin) was added to the cell lysates. Luciferase activity
was measured in a luminometer.
Morphometric Analysis: Interstitial Volume
Measurements of interstitial volume were undertaken
using a point-counting technique as previously de-
scribed (Wright et al, 1996). Ten consecutive, non-
overlapping fields were evaluated in both the cortex
and medulla at 400ϫ on each trichrome-stained sec-
tion using a 10 ϫ 10 grid-imprinted reticle. All points
not contained within tubular cells or lumen, Bowman’s
capsule, or blood vessel were considered interstitial.
This fraction correlated with relative interstitial volume,
and was evaluated in a blinded fashion by two indi-
viduals. Results are expressed as the percentage of
the measured area that represents interstitial space.
Macrophage Identification and Quantification
Expression of macrophages was evaluated using im-
munoperoxidase in paraffin sections, as previously
described (Scherr et al 1999; Wright et al, 1996). We
used a rat-monoclonal antibody to the F4/80 antigen
(Caltag Laboratories, Burlingame, California), a murine
macrophage-specific cellular membrane marker, in
formalin-fixed sections. No specific antigen retrieval
was required using this antibody in paraffin-fixed
sections.
Sections were deparaffinized with Hemo-De for 30
minutes, rehydrated in increasing concentrations of
ethanol, and washed three times in PBS for 10 min-
utes. Endogenous peroxidase was quenched for 30
minutes with 0.3% H O /methanol solution. After
Tissue Hydroxyproline
Hydroxyproline was measured using the colorimetric
assay of Reddy and Enwemeka (1996). This assay
uses Ehrlich’s reagent and chloramine-T and is read at
2
2
5
50 nm. Tissues were lyophilized and results ex-
washes in filtered water and PBS, a blocking step was
included using 5% rabbit serum in conjunction with
the avidin and biotin blocking solutions (Vector Kit;
Vector Laboratories, Burlingame, California) for a total
of 30 minutes. Primary antibody (Ab) was then applied
pressed per mg dry weight of tissue. Hydroxyproline is
considered to represent 10% of the collagen, as
determined by using collagen standards in this assay.
Results are adjusted accordingly.
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726 Laboratory Investigation • November 2000 • Volume 80 • Number 11

Interstitial Fibrosis of UUO in iNOS -/- Mice
(
dilution 1:150) overnight at 4° C. Mouse adsorbed,
biotinylated rabbit anti-rat secondary Ab (Caltag;
:100) was incubated for 30 minutes, as was tertiary
Hierholzer C, Harbrecht B, Menezes JM, Kane J, MacMicking
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1
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1
0% hematoxylin for 2 minutes. Macrophages were
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Ishidoya S, Morrissey J, McCracken R, Reyes A, and Klahr S
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Interstitial volume and macrophage number compari-
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