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*
Department of Internal Medicine, Division of Nephrology and Hypertension, and
Department of Gene Therapy, Institute of DNA Medicine, Jikei University School of Medicine, Tokyo, Japan; and
Department of Internal Medicine, Saitama Medical School, Kawagoe, Saitama, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
IL-1R expression increased within 3 days compared with contralateral
untreated kidneys in the same mice. Similarly, the macrophage
infiltration in the cortical interstitium increased after 3 days in UUO
kidneys, but not untreated kidneys. After UUO developed, DBA/2j mice
were injected i.v. with either IL-1ra+ vehicle cells
(IL-1ra-treated mice) or GC+ vehicle cells (GC-treated
mice) at 24 h after UUO. Six days after the injection of these
vehicle cells, marked increase of CD11b+
IL-1ra+ vehicle cells was observed in the ICAM-1-positive
interstitium of UUO kidneys from IL-1ra-treated mice. In contrast, no
CD11b+ IL-1ra+ cells appeared in
ICAM-1-negative contralateral kidneys from these mice. Furthermore, the
infiltration of macrophages (p < 0.001),
expression of ICAM-1 (p < 0.005), and presence of
-smooth muscle actin (p = 0.005) in the
interstitium of UUO kidneys were significantly decreased in
IL-1ra-treated mice compared with GC-treated mice. These findings
suggest that IL-1 may contribute to the development of renal
interstitial injury and that our method can deliver a functioning gene
encoding an antiinflammatory cytokine gene specifically at that site by
interacting with local adhesion molecules. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-1 is an important proinflammatory cytokine with a wide range of
effects, including activation of endothelial cells, stimulation of
tissue infiltration by neutrophils and macrophages, and induction of
other mediators of inflammation such as TNF-, IL-8, ICAM-1, and NO
(5, 6). As an example of its potential for damage, a
pathological role has been identified for IL-1 in experimental and
human glomerulonephritis (7, 8, 9, 10). Glomerular as well as
tubular epithelial cells may synthesize and release IL-1 (8, 10, 11), yet no proof exists to implicate IL-1 as a cause of
tubulointerstitial injury.
As a modulator of IL-1 activity, the IL-1 receptor antagonist (IL-1ra)3 can suppress IL-1 activity, as evident by the ability of this receptor to block experimental glomerulonephritis (10, 12, 13). Based on this concept, we previously established a novel system for using bone marrow-derived cells as vehicles for site-specific delivery of an IL-1ra gene into inflamed glomeruli. This procedure, which suppressed local IL-1 action (14, 15), successfully prevented the progression of glomerular injury evoked by Ab to the glomerular basement membrane (GBM).
With that background, we initiated this two-part study. For the first part, we examined the time course of macrophage infiltration as well as ICAM-1, IL-1 mRNA, and IL-1R expression in mice treated to develop a unilateral ureteral obstruction (UUO) in the cortical interstitium. This model is a well-established archetype of renal interstitial injury (2, 3, 4). For the second study, we use genetically modified bone marrow-derived CD11b+ CD18+ vehicle cells to deliver IL-1ra to inflamed interstitium of UUO kidneys as a new therapeutic approach for controlling tubulointerstitial injury.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thirty DBA/2j female mice were purchased from Nippon Crea (Tokyo, Japan). All animals used in this study were maintained in our animal facility on standard laboratory chow.
Unilateral ureteral obstruction
At 8 wk of age, 20 mice were anesthetized by the i.p. injection of pentobarbital, and their right ureters were ligated and cut down as described (16) to cause UUO. Five of these mice with UUO kidneys were sacrificed for histological examination and RT-PCR analyses at posttreatment days 3, 5, 7, and 14.
Establishment of vehicle cells
Bone marrow-derived CD11b+ and CD18+ vehicle cells were established as previously described (14). Briefly, bone marrow cells were harvested from the femur and tibia of the 7- to 8-wk-old DBA/2j mice and suspended in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FBS, 20% heat-inactivated horse serum, 20% L-929-conditioned medium, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. Cells were seeded onto unprocessed 10-cm dishes at a concentration of 1 x 107 cells/dish and cultured in a humidified atmosphere of 5% CO2 for 1 wk. These vehicle cells were verified as expressing CD11b and CD18, both of which are ligand of ICAM-1 by FACS (14).
Recombinant adenovirus preparation and in vivo injection of IL-1ra
Replication-defective recombinant adenoviruses carrying IL-1ra
(AxCAmIL-1RA) were purchased from Riken DNA Bank (Ibaraki, Japan),
and adenoviruses carrying glucocerebrosidase (GC) cDNA (AxACGC)
(17) were kindly provided by Dr. T. Ohashi (Department of
Gene Therapy, Jikei University School of Medicine, Tokyo, Japan). Both
were under the control of a CMV enhancer chicken -actin hybrid
promoter (18). Recombinant viruses were propagated and
isolated from 293 host cells. Bone marrow-derived vehicle cells, as
described above, were cultivated with 10% FBS, 20% horse serum, and
20% L-929-conditioned medium for 6 days, and then AxCAmIL-1RA or
AxCAGC was infected at a multiplicity of infection = 200.
Mice with UUO were injected through the tail vein with 5 x 106 of either IL-1ra-infected vehicle cells (IL-1ra-treated group, n = 5), or GC-infected vehicle cells (GC-treated group as a control, n = 5) at 24 h after UUO treatment. Six days after the injection of these vehicle cells, sera from individual mice were collected, and all mice were sacrificed to obtain kidney tissues for histological examination.
Immunohistochemistry
The detection of infiltrating F4/80-positive macrophages,
infused Mac-1+ (CD11b) vehicle cells, and ICAM-1
or -smooth muscle actin (-SMA) expression in the cortical
interstitium relied on immunohistochemistry based on the
avidin-biotin-peroxidase method, as described (19). Kidney
specimens were embedded in OTC compound (Miles Scientific, Naperville,
IL) and quickly frozen in dry ice and acetone at -70°C. Cryostat
sections (3 µm) were rinsed in PBS (10 mmol/L sodium phosphate, pH
7.2, 0.9% saline) three times for 15 min each. First, the endogenous
biotins in the sections were blocked as directed in an
avidin/biotin-blocking kit (Vector Laboratories, Burlingame, CA). Next,
the sections were incubated with the primary Abs overnight at 4°C.
Primary Abs used in this study were rat anti-mouse F4/80 mAb for
macrophage staining
(20) (dilution 1/100; BMA Biomedicals AG, Switzerland), rat anti->murine
ICAM-1 mAb (21) (YN1/1.7.4; kindly provided by Toray
Medical Company, Tokyo, Japan), rat anti-murine Mac-1 mAb
(M1-70.15.11.5.HL; American Type Culture Collection, Manassas, VA), or
mouse anti--SMA mAb (dilution 1:50; Dako, Glostrup, Denmark).
Then the sections were incubated with either biotinylated mouse
anti-rat IgG (
)-chain mAb (MARK-1; Zymed Laboratories, San
Francisco, CA) for F4/80, ICAM-1 and Mac-1 staining or biotinylated
rabbit anti-mouse IgG () Ab (American Quolex, La Mirada, CA) for
-SMA staining. After a 60-min incubation at room temperature,
incubation proceeded with avidin-biotin-peroxidase complex (Vector ABC
Elite staining kit; Vector Laboratories, Burlingame, CA). The
peroxidase was developed with a diaminobenzidine substrate solution
(Peroxidase Substrate kit; Vector Laboratories). Both kits were used
according to the manufacturer’s instructions. Finally, the sections
were counterstained with methyl green.
For indirect immunofluorescence, the sections were stained to identify
IL-1ra+ cells, IL-1R, and ICAM-1 expression in
the tubulointerstitium. To prevent nonspecific binding of avidin and
biotin, an avidin/biotin-blocking kit (Vector Laboratories) was used,
after which sections were incubated with either rat anti-murine
IL-1ra Ab (dilution 1/100; Genzyme-Techne, Cambridge, MA), rat
anti-murine ICAM-1 mAb (YN1/1.7.4), or rabbit anti-mouse IL-1R
type 1 (IL-1Rt1) Ab (dilution 1/50; Santa Cruz Biotechnology, Santa
Cruz, CA) overnight at 4°C, followed by rinsing in PBS. Incubation
for 60 min at 37°C followed with biotinylated mouse anti-rat IgG
()-chain mAb (MARK-1) for IL-1ra and ICAM-1 staining or biotinylated
sheep anti-rabbit IgG Ab (American Quolex) for IL-1Rt1 staining.
After rinsing in PBS, the sections were incubated with either
FITC-labeled avidin (dilution 1/50; Becton Dickinson, San Jose, CA) for
ICAM-1 and IL-1Rt1 staining, or rhodamine-labeled streptavidin
(dilution 1/50; American Quolex) for IL-1ra staining for 60 min at
37°C. Negative controls were performed by replacing the first-step Ab
by incubation buffer only or by isotype-matched Abs. The sections were
again rinsed in PBS, then mounted in p-phenylenediamine
(Sigma, St. Louis, MO)-PBS-glycerine, and observed under a Zeiss
Axiophot fluorescence photomicroscope.
Morphometric analysis
Interstitial F4/80-positive cells. The number of F4/80-positive cells in the cortical interstitium was counted using an eyepiece graticule. Twenty microscopic fields were counted, and the average number of F4/80-positive cells within a 1-mm2 cortical area was calculated. Only cells with clearly identifiable nuclei were counted.
Interstitial IL-1ra-positive cells. The number of IL-1ra-positive cells in the cortical interstitium was counted under a high power field (hpf) (x1000) fluorescence microscope. The value shown for each group represents an average number of IL-1ra-positive cells obtained from a series of randomly selected fields (more than 10) in each section.
Interstitial ICAM-1 expression. The degree of ICAM-1 expression in the cortical interstitium was determined as a percentage of cortical ICAM-1-positive interstitial area over the total cortical interstitial area among more than 20 fields in each section of the kidney. Each field was scored from 0 to 4: 0 = no changes; 1 = changes affecting <25% of total interstitial area; 2 = changes affecting 25% to 50% of the total interstitial area; 3 = changes affecting 50% to 75% of the total interstitial area; 4 = changes affecting >75% of the total interstitial area. A mean value was calculated to represent the degree of these changes for each section.
Interstitial -SMA expression.
Interstitial immunostaining for -SMA was quantified by the pointing
counting method (22) using an eyepiece graticule of 1
cm2 with 10 equidistant lines at a final
magnification of x400. The results were expressed as the percentage of
-SMA-positive area in the cortical interstitium calculated according
to the following formula and averaged among more than 20 fields in each
section. The percentage of -SMA-positive area = (number of grid
intersections with -SMA-positive staining in the
interstitium)/(total number of grid intersections in the
interstitium) x 100. Interstitium was defined as that portion of
the cortex excluding glomeruli, tubules, arteries, arterioles, and
veins. Fields of medulla were not included in this study.
RT-PCR and preparation of the mouse IL-1 cDNA probe
The renal cortex was isolated from individual mice in all groups stored at 4°C and used for mRNA extraction with a QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia Biotech AB, Uppsala, Sweden).
First-stranded cDNA was synthesized using avian myeloblastosis virus
reverse transcriptase (Roche, Germany). Five microliters of each cDNA
were amplified to a final volume of 20 µl PCR mixture containing 10
mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 200 µM
each of dNTP, 10 pmol of oligonucleotide primers for either IL-1 or
GAPDH, and 0.5 U of Taq DNA polymerase (TaKaRa Shuzo, Kyoto,
Japan). PCR was conducted for 29 cycles for IL-1 or 23 cycles for
GAPDH with a thermal cycler (PC-701; ASTEC, Fukuoka, Japan), as
follows: 94°C for 30 s, 58°C for 30 s, and 72°C for
30 s. The sequence of primers is as follows: IL-1: (sense)
5'-TTGAAGAAGAGCCCATCCTC-3', (antisense)
5'-GAGGTGCTGGATGTACCAGTT-3', and GAPDH: (sense)
5'-AAGGTCATCCATGACAACTT-3', (antisense) 5'-CAGTGTAGCCCAGGATGCC-3',
respectively. The predicted sizes of PCR products for IL-1 and GAPDH
are 411 and 348 bp, respectively.
For semiquantitative analysis, each PCR product was visualized with ethidium bromide after agarose gel electrophoresis. The intensity of fluorescence for each PCR product was calculated with National Institutes of Health image 1.58 software, and normalized by that of GAPDH mRNA. DNA markers (100-bp DNA ladder) were purchased from New England Biolabs (Beverly, MA).
Analysis of serum creatinine
Concentrations of serum creatinine were measured using the VISION analysis kit (Abbott Laboratories, North Chicago, IL), which is based on the Jaffe reaction.
Statistical analysis
Results are expressed as means ± SD. Statistical analysis was performed using the two-sample t test to compare data in different groups and repeated measure ANOVA to compare scores of ICAM-1 expression and numbers of F4/80-positive macrophages. A p value of less than 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first investigated ICAM-1 expression and the infiltration of
macrophages into kidney sections of test mice. At day 3 after UUO,
ICAM-1 was clearly observed by immunostaining of cortical tubular
epithelial cells, interstitia, and vessels of treated kidneys, whereas
contralateral untreated kidneys contained no apparent ICAM-1 (Fig. 1
, A and B). In
addition, an abundance of F4/80-positive macrophages was present in the
interstitium of UUO kidneys (Fig. 2). As
shown in Fig. 3, interstitial expression
of ICAM-1 in UUO kidneys peaked at day 5, and ICAM-1 expression
significantly increased throughout the observation period (mean score:
day 3, 0.5 ± 0.1; day 5, 1.8 ± 0.2; day 7, 1.9 ± 0.4;
day 14, 1.9 ± 0.4, p < 0.0001). The number of
F4/80-positive macrophages in the interstitium of UUO kidneys clearly
correlated with ICAM-1 expression and significantly increased with time
starting at 3 days, peaking at 7 days, and remaining stable until 14
days after UUO developed (day 3, 59.3 ±
13/mm2; day 5, 162.4 ±
16.5/mm2; day 7, 244.9 ±
17.4/mm2; day 14, 236.5 ±
25/mm2, p < 0.0001 in Fig. 3).
No such changes were found in the contralateral untreated kidneys
throughout the observation period. Thus, ICAM-1 expression may recruit
macrophages into the interstitium of UUO kidneys.
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and IL-1 receptor expression in UUO
kidneys
Next we used RT-PCR to examine IL-1 expression in the renal
cortexes of mice with UUO at posttreatment day 3 (Fig. 4). UUO kidneys showed a significant
4-fold increase of IL-1 mRNA compared with contralateral untreated
kidneys. Immunofluorescence revealed IL-1Rt1 receptor weakly expressed
at the apical membranes of tubules and vascular endothelial cells in
untreated kidneys, whereas IL-1R expression was up-regulated in both
the luminal and basolateral membranes of tubules from UUO kidneys (Fig. 5). This heightened expression of IL-1R
and also of IL-1 mRNA endured in UUO kidneys up to day 14.
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We previously established bone marrow-derived vehicle cells that
express the ICAM-1 ligands, CD11b and CD18, and adenovirally transduced
these cells with a gene encoding IL-1ra (IL-1ra+
vehicle cells) (14, 15). Western blot analysis confirmed
that these vehicle cells synthesized and secreted IL-1ra protein
(15). To examine the function of these cells in the UUO
system, CD11b+ IL-1ra+
vehicle cells were injected i.v. into mice with UUO. Six days later,
CD11b+ cells were clearly visible in the
interstitium of UUO kidneys expressing ICAM-1, but not ICAM-1-negative
untreated kidneys (data not shown). Immunohistochemical study (Fig. 6, A and B)
revealed significant numbers of vehicle cells producing IL-1ra within
the interstitium of UUO kidneys from IL-1ra-treated mice, whereas few
IL-1ra+ vehicle cells occupied in the
interstitium of UUO kidneys from GC-treated mice (the number of
IL-1ra+ vehicle cells per hpf: 7.2 ± 0.8 vs
0.8 ± 0.4, respectively, p < 0.0005). Parts of
cortical tubules in UUO kidneys were also stained with IL-1ra. In
contrast, IL-1ra+ vehicle cells were not observed
in contralateral kidneys from either IL-1ra-treated mice or GC-treated
mice (the number of IL-1ra+ vehicle cells per
hpf: 0.6 ± 0.4 vs 0.4 ± 0.2, respectively). Apparently,
infused CD11b+ IL-1ra+
vehicle cells site specifically delivered IL-1ra into the inflamed
interstitium by interaction with ICAM-1.
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To investigate the physiological effects of
IL-1ra+ vehicle cells, we next measured serum
creatinine levels and assessed renal histology. Six days after the
injection of either IL-1ra+ vehicle cells or
GC+ vehicle cells, serum creatinine levels of
both recipient groups were similar: IL-1ra-treated mice, 0.48 ±
0.21 mg/dl vs GC-treated mice, 0.58 ± 0.22 mg/dl
(p > 0.05) (data not illustrated). After
histological analysis (Fig. 7A), interstitial ICAM-1
staining of UUO kidneys was significantly lower in IL-1ra-treated mice
than in GC-treated mice (mean scores, respectively: 1.5 ± 0.3 vs
2.1 ± 0.1, p < 0.005). To the contrary, the
interstitium of contralateral kidneys from IL-1ra-treated mice and
GC-treated mice had only faint immunostaining for ICAM-1 (mean score:
0.2 ± 0.1 and 0.3 ± 0.1, respectively). In addition,
IL-1ra-treated mice had a markedly decreased number of macrophages in
the interstitium of their UUO kidneys compared with GC-treated mice
(191.1 ± 17.9/mm2 vs 343.4 ±
8.13/mm2, p < 0.001 in Fig. 7B). However, similar numbers of interstitial macrophages
were observed in contralateral kidneys of IL-1ra- and GC-treated mice
(41.5 ± 11.5/mm2 vs 25.5 ±
8.70/mm2, respectively).
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-SMA may be an important factor in the interstitial fibrosis
(23, 24), we next injected IL-1ra+
vehicle cells to assess the effect on interstitial expression of
-SMA in UUO kidneys. As Fig. 8A illustrates, intense and
widespread -SMA immunoreactivity developed in the interstitium of
UUO kidneys from GC-treated mice. However, IL-1ra-treated mice had
significantly less interstitial -SMA expression in UUO kidneys (Fig. 8B). Fig. 9 shows that the
percentage of -SMA-positive area in total interstitial area of each
UUO kidney was significantly decreased in IL-1ra-treated mice compared
with GC-treated mice (27.4 ± 2.7 and 36.3 ± 4.4,
respectively, p = 0.005). In contrast, no interstitial
-SMA was detectable in the untreated kidneys of either
IL-1ra-treated mice or GC-treated mice (percentage of -SMA-positive
area in total interstitial area: 1.7 ± 1.1 and 2 ± 0.6,
respectively). IL-1ra+ vehicle cells may then
suppress both interstitial ICAM-1 expression and the macrophage
infiltration, thereby inhibiting -SMA expression in UUO kidneys.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Interstitial ICAM-1 was expressed within 3 days after treatment to
induce UUO, with a peak response by the fifth day that stayed high up
to the fourteenth day. Subsequently, macrophages were recruited into
the interstitium in these UUO kidneys (Figs. 2 and 3). In addition,
interstitial expression of ICAM-1 was most intense in areas of marked
macrophage infiltration. Numerous studies have indicated that ICAM-1
plays a major role in immune injury to the kidney (25, 26). In particular, Ricardo et al. (27)
demonstrated that ICAM-1 on the renal cortex is important for
recruiting macrophage into the interstitium of obstructed kidneys,
supporting our observations. Furthermore, Cheng et al.
(28) reported that treatment with ICAM-1 antisense
oligonucleotides alleviated the infiltration of inflammatory cells and
accumulation of the extracellular matrix in the tubulointerstitium of
mice with renal UUO.
The stimulus that induces ICAM-1 to appear tubular epithelial cells and
interstitial cells in the renal cortex after ureteral ligation remains
unclear. However, proinflammatory cytokines such as IL-1, TNF-, and
IFN-
are considered inducers of ICAM-1 expression on glomerular
endothelial cells, mesangial cells, and renal tubular epithelial cells
(29, 30). Recently, we and others found that blocking the
action of IL-1 by the administration of IL-1ra prevented renal injury
in a rat model of anti-GBM glomerulonephritis (7, 8, 9, 10, 11, 12, 15). Thus, our attention focused on the role of IL-1 in the
development of tubulointerstitial injury after the onset of renal
UUO.
Our former PCR analysis showed increased levels of IL-1 mRNA in
kidneys with induced UUO, suggesting that IL-1 may be synthesized in
the renal cortex. Although we could not identify the origin of IL-1
in acetone-fixed tissues from UUO kidneys, tubular IL-1 expression
in glomerulonephritic humans and animals has been demonstrated with
PLP-fixed tissues or in situ hybridization (8, 10, 13). In
the present study, the expression of IL-1Rt1 receptor in the tubules
was up-regulated in the UUO kidneys. These data suggested that IL-1 may
enhance ICAM-1 expression on tubular epithelial cells and interstitial
cells via both autocrine and paracrine modes.
Currently, no clinical therapy effectively halts the interstitial
fibrosis of progressive renal diseases. However, local delivery of
anti-inflammatory cytokines using gene transfer may provide such a
tool. To adapt that technology, we established a novel ex vivo gene
delivery system that uses bone marrow-derived vehicle cells bearing
CD11b and CD18; because these molecules are ligands of ICAM-1, they
promote the cells’ migration to sites of ICAM-1 expression
(14). Using this system, we delivered IL-1ra into
ICAM-1-expressing inflamed glomeruli in rabbits with anti-GBM
glomerulonephritis and blocked the progression of glomerular injury by
local suppression of IL-1 action (15). In our preliminary
examinations, infused vehicle cells were recruited into the
interstitium of UUO kidneys at 3 days and remained stable until 14 days
after UUO developed. Next, we used these vehicle cells expressing CD11b
and CD18 to deliver IL-1ra into inflamed renal interstitium through
site-specific interaction with ICAM-1 in an attempt to attenuate
interstitial injury in mice induced to develop UUO. Although
interstitial ICAM-1 was expressed by the third day after treatment to
incite UUO in our study, others demonstrated increased interstitial
ICAM-1 expression in the renal cortex by 12 h after the inception
of UUO (28, 32). Therefore, our mice were injected i.v.
with syngeneic bone marrow-derived vehicle cells at 24 h after UUO
induction. Immunohistochemical studies later confirmed that infused
CD11b+ cells producing IL-1ra were recruited into
the cortical interstitium of UUO kidneys. Moreover,
CD11b+ cell recruitment correlated with ICAM-1
expression. In contrast, parts of cortical tubules in UUO kidneys were
stained with IL-1ra, suggesting that intracellular form of IL-1ra might
be expressed in tubular epithelial cells as in keratinocytes and
intestinal epithelial cells (33). Although IL-1ra is
produced by activated neutrophils and monocytes (31), few
cells producing IL-1ra were recruited into the same animals,
contralateral untreated kidneys, and the kidneys from GC-treated mice.
These data indicated that CD11b+ vehicle cells
can site specifically deliver IL-1ra into inflamed interstitium through
the interaction of adhesion molecules. In addition, IL-1ra secreted by
vehicle cells appeared to suppress IL-1 action and subsequently
decrease tubulointerstitial inflammation by inhibiting ICAM-1
expression and macrophage infiltration into UUO kidneys. In fact,
degrees of tubular expression of IL-1Rt1 receptor did not differ
between IL-1ra-treated group and GC-treated group, and supernatants
from IL-1ra-infected vehicle cells suppress IL-1-induced
PGE2 production in NIH3T3 cells (unpublished
data), suggesting that IL-1ra secreted from IL-1ra-infected vehicle
cells may inhibit the bioaction of IL-1. Although the fate of these
infused vehicle cells is not fully understood, it seems that the
majority stay in the spleen and liver without attaching to adhesion
molecules and cells, but those cells that escape to the circulation
later accumulate at their destined site when adhesion molecules are
expressed (14).
The present study also demonstrated that IL-1ra suppressed the
expression of -SMA in interstitial myofibroblasts, an event that may
influence the process of interstitial fibrosis (24, 25).
IL-ra may use any of several possible pathways to accomplish the
results witnessed in this study. First, IL-ra may blockade the action
of IL-1 on interstitial fibroblasts. IL-1 production is generally
thought to stem from tubular epithelial cells and macrophages
infiltrating UUO kidneys. However, Rubbia-Brandt et al.
(34) demonstrated that s.c. administration of IL-1 into
rat connective tissue did not increase the animals’ content of
-SMA-positive myofibroblasts. Yet, Nikolic-Paterson et al.
(13) found that the suppression of experimental
glomerulonephritis by IL-1ra was mediated by inhibition of ICAM-1
expression. In our study, interstitial ICAM-1 expression was decreased
in UUO kidneys from IL-1ra-treated mice. Furthermore, the IL-1ra
reduced macrophage infiltration and might have suppressed their
function, which would enhance the phenotype change of interstitial
fibroblasts into myofibroblasts (16, 35). Therefore,
IL-1ra treatment may attenuate interstitial fibrosis in obstructed
nephropathy by inhibition of ICAM-1 expression and of macrophage
infiltration.
Systemic administration of IL-1ra has been used as a therapeutic agent for several inflammatory disorders, including glomerulonephritis (10, 11, 12). However, the continuous infusion of high-dose IL-1ra required to ubiquitously block IL-1 action affects normal body functions. Consequently, the local suppression of IL-1 action at specific sites is a more effective mode of therapy for humans. Additionally, our system has an advantage over previous gene delivery methods (28, 36) in the site-specific delivery of a functioning gene that encodes anti-inflammatory cytokine directly into inflamed interstitium through the interaction of adhesion molecules.
In conclusion, this study suggests that IL-1 may contribute to the development of tubulointerstitial injury, and that the delivery of IL-1ra gene by CD11b+ vehicle cells can attenuate interstitial inflammation and fibrosis in obstructive nephropathy. Our novel gene delivery system offers a promising strategy for the treatment of some progressive kidney diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yasunori Utsunomiya, Department of Internal Medicine, Division of Nephrology and Hypertension, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo, 105-8461 Japan.
3 Abbreviations used in this paper: IL-1ra, IL-1 receptor antagonist; GBM, glomerular basement membrane; GC, glucocerebrosidase; hpf, high power field; IL-1Rt1, IL-1R type I; -SMA, -smooth muscle actin; UUO, unilateral ureteral obstruction.
Received for publication June 9, 2000. Accepted for publication October 6, 2000.
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S. Klahr and J. Morrissey Obstructive nephropathy and renal fibrosis Am J Physiol Renal Physiol, November 1, 2002; 283(5): F861 - F875. [Abstract] [Full Text] [PDF]
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), ICAM-1 expression; (
, 
), F4/80-positive cells.
