1 Renal Division, Departments of Medicine, 2 Cell Biology, and 3 Pathology, Barnes-Jewish Hospital at Washington University, St. Louis, Missouri 63110; and 4 Creative Biomolecules, Hopkinton, Massachusetts 01748
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ABSTRACT |
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Unilateral ureteral obstruction (UUO) is a model of renal injury characterized by progressive tubulointerstitial fibrosis and renal damage, while relatively sparing the glomerulus and not producing hypertension or abnormalities in lipid metabolism. Tubulointerstitial fibrosis is a major component of several kidney diseases associated with the progression to end-stage renal failure. Here we report that when a critical renal developmental morphogen, osteogenic protein-1 (OP-1; 100 or 300 µg/kg body wt), is administered at the time of UUO and every other day thereafter, interstitial inflammation and fibrogenesis are prevented, leading to preservation of renal function during the first 5 days after obstruction. Compared with angiotensin-converting enzyme inhibition with enalapril treatment, OP-1 was more effective in preventing tubulointerstitial fibrosis and in preserving renal function. The mechanism of OP-1- induced renal protection was associated with prevention of tubular atrophy, an effect not shared with enalapril, and was related to preservation of tubular epithelial integrity. OP-1 blocked the stimulation of epithelial cell apoptosis produced by UUO, which promoted maintenance of tubular epithelial integrity. OP-1 preserved renal blood flow (RBF) during UUO, but enalapril also stimulated RBF. Thus OP-1 treatment inhibited tubular epithelial disruption stimulated by the renal injury of UUO, preventing tubular atrophy and diminishing the activation of tubulointerstitial inflammation and fibrosis and preserving renal function.
kidney morphogens; tubulointerstitial fibrosis; renal failure; tubular atrophy
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INTRODUCTION |
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BONE MORPHOGENETIC
PROTEIN-7 (BMP-7), also known as osteogenic protein-1 (OP-1), a
member of the transforming growth factor- (TGF-
) superfamily, is
a key morphogenic signal for kidney development (36). OP-1
(21, 37, 48, 51)
continues to be expressed in adult mammals predominantly within the
kidney (45, 47) and can be found in the
circulation (Sampath TK, unpublished observations). Deletion of OP-1 in
mice results in failure of differentiation of the metanephric
mesenchyme, leading to loss of mesenchymal cell condensation around the
ureteric bud and eventually to glomerular agenesis (15,
36, 51). OP-1-deficient animals die shortly after birth due to uremia. Many features of development are
recapitulated during renal injury, and OP-1 may be important in both
preservation of function and resistance to injury (50).
OP-1 has been shown to decrease the loss of kidney function associated
with acute ischemic injury (50). Although the function of
OP-1 in the kidney and in renal injury is unknown, it may have a
cytoprotective effect, or it may regulate chemotactic cytokines
involved in monocyte infiltration associated with a variety of renal
diseases. We hypothesize that OP-1, in addition to its role as a renal
morphogen, is a critical homeostatic factor preserving renal function,
and here we report the importance of OP-1 in preventing
tubulointerstitial fibrosis.
To simplify the complex milieu of renal diseases associated with
interstitial fibrosis, we utilized the unilateral ureteral obstruction
(UUO) model of renal injury (41). In this model, hypertension, proteinuria, and lipid dysregulation do not contribute to
progressive nephron destruction (6, 16,
20, 26, 32), and glomerular
injury is not prominent early in the course of the injury produced.
Uremia is avoided by the function of the contralateral kidney, which
undergoes hypertrophy and hyperplasia as the obstructed kidney is
destroyed. The renal injury of UUO is mediated in part through
stimulation of renal angiotensin II production, which activates TGF-
in a cascade of events culminating in tubulointerstitial inflammation
and fibrosis (13, 17, 18, 34, 52). Inhibition of angiotensin II
production by angiotensin-converting enzyme (ACE) inhibitors decreases
expansion of the renal interstitium associated with fibrosis
(23, 31). We have also shown that ACE
inhibitors decrease transformation of renal cells to interstitial myofibroblasts and diminished infiltration of the interstitial compartment by inflammatory cells (23, 31).
However, limiting angiotensin II production through direct modulation
of angiotensinogen expression does not attenuate tubular atrophy
(18). Here, we demonstrate that OP-1 was more effective
than ACE inhibition in preserving renal structure and function in rats
with UUO. OP-1 administration preserved tubular integrity by preventing
epithelial cell apoptosis. OP-1 treatment prevented the invasion of
renal parenchyma by mononuclear phagocytic cells and the expansion
of extracellular matrix in the renal interstitium to a greater extent than did enalapril treatment. Although UUO diminished OP-1 expression in the obstructed kidney, OP-1 administration protected its own expression, perhaps reflecting preservation of tubular epithelial integrity. As a result, tubular atrophy was diminished. These studies
provide a basis for the consideration of OP-1 as a therapeutic candidate for renal protection against tubulointerstitial fibrosis.
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METHODS |
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Preparation of animals.
Sprague-Dawley rats (250 g) underwent either a sham operation (ureter
manipulated but not ligated) or unilateral ureteral ligation. Two
ligatures, 5 mm apart, were placed in the upper two-thirds of the
ureter over a section of polyethylene tubing placed around the ureter.
Ureteral obstruction was confirmed by observation of dilation of the
pelvis and proximal ureter and collapse of the distal ureter. The
suture tied to obstruct the ureter was removed along with the tubing at
day 5, relieving the obstruction. Urine cultures obtained at
time of release were negative for bacteria. The following groups were
studied at day 10: sham-operated animals; animals with
release of ureteral obstruction at 5 days; and animals with sustained
UUO for 10 days. Sham-operated rats and rats with UUO of 5 days
duration were studied by inulin and p-aminohippurate (PAH)
clearances at day 10. The rats received one of the following
10 min before the UUO: soluble OP-1 (100 µg/kg body wt ip every other
day; n = 6); soluble OP-1 (300 µg/kg body wt ip every
other day; n = 6); enalapril (~25 mg · kg body wt1 · day
1 ingested) in the drinking
water (n = 6); or vehicle (n = 6). The
same experimental protocol was used in rats with UUO of 10 days
duration (n = 6 in each of 4 groups).
Preparation of OP-1. The full-length human OP-1 cDNA (46) was expressed in Chinese hamster ovary cells and purified from the medium as a soluble complex. The complex was composed of the processed mature BMP-7 homodimer noncovalently attached to prodomain protein (referred to as soluble OP-1) (27).
Preparation of kidneys.
After completing the clearance studies, the animals were euthanized
(methoxyflurane anesthesia), and the kidneys were thoroughly perfused
with ice-cold Hanks' balanced salt solution (HBSS) to remove
blood-borne cells. Kidneys were rapidly removed and sliced on a cold
glass plate. For histological studies, 2-mm coronal sections of the
HBSS-perfused kidneys were immersed in Histochoice (Amresco, Solon, OH)
or in buffered Formalin. Kidney sections were embedded in paraffin, and
5-µm sections were analyzed microscopically. We evaluated renal
fibrosis by determining interstitial collagen deposition and measuring
interstitial volume, immunostaining for type IV collagen,
immunostaining for -smooth muscle actin (
-SMA), and by
quantitating monocyte/macrophage infiltration, as previously reported
(23, 24, 31, 40).
Quantitation of fibrosis. Interstitial volume was determined by a point-counting technique on tissue sections stained by the Masson's Trichrome method and was expressed as the mean percentage of grid points lying within the interstitial area in up to five fields in the cortex. A 1-mm2 graded ocular grid viewed at ×200 magnification delineated each of these fields. Morphometric data were plotted against the experimental durations and expressed as means ± SD. Statistical difference was assessed by analysis of variance. P < 0.05 was considered to be significant.
The degree of fibrosis or the potential for fibrosis in the kidney was determined by scoring the amount of interstitial collagen IV andQuantitation of monocyte/macrophage interstitial infiltration.
Counting ED-1 antigen-positive cells in sections of obstructed and
nonobstructed kidneys quantitated the infiltration of the renal
parenchyma by cells of the monocyte/macrophage lineage. Kidneys were
fixed in Histochoice, paraffin embedded, and sectioned. Sections were
dewaxed and rehydrated before incubation with the ED-1 antibody
obtained from Harlan (Indianapolis, IN). A fluorescent-labeled second
antibody was used to develop staining of positive cells. Five
consecutive nonoverlapping fields were counted in three sections from
five to seven animals and viewed at ×200
magnification.
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Characterization of OP-1 expression. OP-1 expression was analyzed in 5-µm-thick rat kidney sections fixed in Histochoice and embedded in paraffin. Immediately before staining, tissue sections were dewaxed in a series of solvent baths and then rinsed three times with PBS. Potential sites for nonspecific antibody binding were blocked by a 30-min incubation with 2% BSA, and 0.04% sodium azide in PBS at room temperature. The primary antibody, a protein A column-purified mature OP-1 polyclonal antibody raised in sheep, was diluted 1:500 in 2% BSA and 0.04% sodium azide in PBS, resulting in antibody concentration of ~7 µg/ml, and incubated with the sample for 1 h at room temperature. This was followed by four 15-min washes with PBS and a 1-h incubation with a secondary indocarbocyanine-conjugated anti-sheep IgG (1:200 dilution in 2% BSA and 0.04% sodium azide in PBS). Cells were washed as before with PBS and mounted on glass microscope slides with Aqua Polymount. Results were analyzed on a Zeiss fluorescent microscope (Axioskope) by using ×20 and ×40 objectives, as well as a ×63 oil-immersion objective.
Quantitation of tubular atrophy. Kidney sections were stained with periodic acid Schiff (PAS) for assessment of tubular basement membranes, and tubular atrophy was determined as described by Chevalier et al. (8). Atrophic tubules were identified by their thickened and sometimes duplicated basement membranes. The number of atrophic tubules per field of a ×40 objective was counted, and 50 fields/kidney section were analyzed.
Quantitation of tubular cell apoptosis. Buffered Formalin-fixed sections were deparafinized, rehydrated, and subjected to a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The commercially available T&T TUNEL-like in situ nonisotopic method (Amersham Pharmacia Biotech, Piscataway, NJ) was used as described by the manufacturer, except for reducing the volumes of reagents applied to the slides. The number of TUNEL-positive cells in five nonoverlapping fields of cortex in each kidney section was averaged for the value of the individual kidneys. The average of each of four to six separate kidneys was, in turn, averaged for the mean ± SD for each condition.
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RESULTS |
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We analyzed the degree of tubulointerstitial fibrosis after UUO in
cortical sections of rat kidneys subjected to either 5 or 10 days of
UUO, as described in METHODS. Masson's Trichrome-stained sections were used to analyze the accumulation of collagen in the
interstitium. With this stain, collagen is colored blue and cells are
red. As shown in Fig. 1, collagen was detected in Bowman's capsule of
normal kidneys, the perivascular adventium (Fig. 1A), and in
the renal capsule (Fig. 1B). In comparison, kidneys
subjected to UUO for 5 days with release and recovery for 5 days before analysis at day 10 had massive accumulation of collagen in
the expanded interstitium along with cellular interstitial infiltrates (Fig. 1, C and D). Contralateral, nonligated
kidneys in all of the experimental groups were indistinguishable from
normal kidneys (Fig. 2). Kidneys from
rats given 100 µg/kg of OP-1 with 5 days of UUO before release of the
obstruction and examination at day 10 had minimal
interstitial collagen deposition (Fig. 1, E and F). Rats treated with 300 µg/kg of OP-1 had almost no
renal interstitial collagen deposition after UUO (Fig. 1, G
and H). Expansion of the interstitial space after UUO is
apparent in Fig. 1 (C and D), and this was
quantitated as described in METHODS. UUO with release at 5 days and analysis at 10 days revealed a fivefold expansion of the
interstitial space from 9.7 to 47.4% of the cortical volume (Fig.
3). In the group of rats given 300 µg/kg of OP-1, the expansion of the interstitial space was only
17.1% (<2-fold above normal). Administration of 300 µg/kg of OP-1
was significantly more efficacious than the administration of 100 µg/kg of OP-1 (P < 0.03) (Fig. 3). Treatment with
OP-1 at either dose afforded greater protection than enalapril
(P < 0.01), which we have previously shown to be renal
protective in this model of fibrogenesis (23, 31).
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The expansion of the renal interstitium after UUO was further examined
by analyzing the deposition of type IV collagen. Type IV collagen is a
normal component of tubular basement membranes, which frequently abut
basement membranes of neighboring tubules in normal and contralateral
kidneys (Fig. 4, A and
B, respectively). In obstructed kidneys, expansion of the
interstitial compartment increases the distance between tubular
basement membranes (Fig. 4, C and D). Type IV
collagen staining of the tubular basement membrane was decreased in UUO
and was aberrantly expressed as a component of the interstitial
collagen accumulation. The interstitial myofibroblast responsible for
deposition of the increased interstitial collagen matrix in
tubulointerstitial fibrosis is known to secrete type IV collagen
(2). Thus type IV collagen staining serves as both a
measure of interstitial volume and a marker of interstitial collagen
accumulation. Treatment with OP-1 diminished the distance between
tubular basement membranes, confirming the interstitial volumetric
measurements shown in Fig. 3, and decreased the deposition of type IV
collagen in the interstitium. (Fig. 4, E-H: E and
F, 100 µg/kg OP-1; G and H, 300 µg/kg OP-1 and Fig. 5).
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We next examined the expression of -SMA, a marker of the
tubulointerstitial myofibroblast responsible for a large component of
the interstitial collagen deposition after UUO. In the normal and
contralateral kidneys,
-SMA was expressed only in the blood vessels
(Fig. 6, A and B),
but it was prominently expressed in the interstitium of the kidneys
subjected to 5 days of UUO and released before analysis at day
10 (Fig. 6C). OP-1 administration significantly reduced
interstitial
-SMA (Fig. 6D), as demonstrated by a lesser
histological scoring of
-SMA (Fig. 6E). The effect of
OP-1 on
-SMA histological scores was dose dependent, and the 300 µg/kg dose was significantly more effective than enalapril in
preventing accumulation of cells expressing
-SMA in the
interstitium.
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OP-1 treatment also prevented the infiltration of the renal parenchyma
by macrophages after UUO (13). Using a monoclonal antibody
(ED-1) to a macrophage-specific antigen as described in
METHODS, we quantitated the cellular infiltration of the
interstitium after UUO. Administration of OP-1 at 100 or 300 µg/kg
body wt reduced the macrophage infiltration by 55 and 72%,
respectively, in the UUO kidneys from rats whose obstruction was
released at day 5 after analysis at day 10 (Fig.
7). Although enalapril also reduced
macrophage infiltration (Fig. 7), the protective effect of 300 µg/kg
OP-1 every other day was significantly greater than that of enalapril.
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To assess the potential effect of OP-1 on renal function, we performed
renal clearance studies in each of the kidneys of rats after 5 days of
UUO and 5 days of recovery. As shown in Fig.
8, there was no recovery of renal
function in the postobstructed kidneys of vehicle- or enalapril-treated
animals. However, the postobstructed kidneys of 50% of each of the
OP-1-treated groups had return of urine flow after release of UUO, and
the mean GFR in the animals from the 100 and 300 µg/kg groups was
0.19 and 0.21 ml · min1 · 100 g body
wt
1, or 34 and 38% of normal GFR values, respectively
(Fig. 8B). The GFR of the contralateral kidneys tended to be
increased in the enalapril-treated group similar to that of the vehicle
group, compared with sham, suggesting compensatory hypertrophy (Fig. 8B), and contralateral renal hypertrophy was less in the
OP-1-treated groups. RBF was assessed by PAH clearances, and as a
result, assessment was limited to those animals with return of urine
flow. Mean single-kidney PAH clearances were 1.0 and 1.2 ml · min
1 · 100 g body wt
1 in the 100 and
300 µg/kg body wt OP-1 groups, respectively, or 58 and 68% of the
values obtained in sham-operated normal rats (Fig. 8C). RBF
in the contralateral kidneys was not altered, except for a modest but
significant (P < 0.05) increase in the
enalapril-treated group.
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We also analyzed the protective action of OP-1 during a longer period
of UUO sufficient to produce a severe injury that would be irreversible
in the experience of most investigators. As shown in Fig.
9, 10 days of sustained UUO in
vehicle-treated rats resulted in a further increase in interstitial
volume from 47.4% in the 5-day obstruction/5-day release group to a
level of 57.2%. This increase, while significant, represents some
leveling off in the rate of interstitial volume increase. Concordant
with this observation, there was no further induction of -SMA or
increase in the interstitial type IV collagen score when values from
the release at 5 days are compared with those from the 10-day
sustained-UUO animals. OP-1 treatment, especially using the 300 µg/kg
body wt dose, significantly decreased interstitial volume, the collagen
IV matrix score, and the expression of
-SMA (Fig. 9,
A-C). Visual inspection of the immunocytochemistry for
collagen IV and
-SMA (not shown) confirmed the scoring results. In
rats with sustained UUO for 10 days, administration of OP-1 reduced the
cellular infiltration (Fig. 9D). The administration of OP-1,
especially the 300 µg/kg dose, afforded greater protection than
enalapril administration in the rats with UUO of 10 days duration (Fig.
9).
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To assess the mechanism of protection by OP-1 against UUO-stimulated tubulointerstitial fibrosis, we first examined the effects of UUO on tubular atrophy.
We quantitated tubular atrophy as described by Chevalier et al.
(8). UUO of 5 days duration, with 5 days of recovery and analysis at day 10, significantly increased the number of
atrophic tubules (Fig. 10, B
and F). OP-1 dose dependently decreased tubular atrophy
(Fig. 10, C, D, and F). Enalapril had
no effect on the tubular atrophy produced by UUO, in agreement with
other studies (18) (Fig. 10, E and
F). These data demonstrate a significant difference between
the mechanism of renal protection in a comparison of OP-1 and enalapril
treatment. They suggest that the mechanism of OP-1-induced renal
protection was related to preservation of renal tubular epithelial
integrity and that this may have served to suppress the
tubulointerstitial inflammation and fibrosis due to the UUO-stimulated,
angiotensin II-mediated damage cascade.
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To further investigate the mechanism of renal tubular epithelial
preservation after UUO, we examined whether apoptotic loss of
epithelial cells, which has been shown to contribute to tubular atrophy
and to be increased by UUO (49), was affected by OP-1 treatment. As shown in Fig. 11,
sections of kidney, similar to those seen in Fig. 10, were examined by
the TUNEL assay for apoptotic nuclei. The overall results are
summarized in Fig. 11. In vehicle-treated rats that had undergone 5 days of UUO with analysis on day 10, a significant
(P < 0.001) increase in the number of apoptotic nuclei
of epithelial cells was found, from an average of 1.7/×200 microscopic
field in the kidney of sham-operated animals (Fig. 11A) to
30.1 apoptotic nuclei/×200 field (Fig. 11B). Both doses of
OP-1 significantly reduced the number of apoptotic nuclei by about
one-third (P < 0.001), as seen in Fig. 11,
C and D. Enalapril produced a modest but
significant (P < 0.05) decrease of ~10% in tubular
epithelial cell apoptosis (Fig. 11E).
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Tubular epithelial morphology in the medulla was preserved by
OP-1 treatment, as shown in Fig. 12.
UUO for 5 days with recovery for 5 days after release was characterized
by a flattened, effaced epithelia in medullary tubules, and the OP-1
dose dependently preserved epithelial morphology. Associated with
preservation of epithelial cell morphology, expression of OP-1 was
protected. OP-1 is expressed in the collecting duct epithelia
(45, 47), and its expression was preserved by
OP-1 treatment but not by enalapril (Fig. 12). Because the half-life of
OP-1 is short and the last dose was administered 48 h before
analysis, exogenous OP-1 was not detected by immunostaining.
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DISCUSSION |
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We show here that systemic administration of human recombinant
OP-1 to rats with UUO produced nearly complete protection for 5 days
against tubulointerstitial fibrosis. Tubulointerstitial fibrosis is a
common final pathway contributing to progression of many chronic kidney
diseases (11, 12, 26). UUO
activates a cascade of events that produce tubulointerstitial fibrosis
(18, 32, 34). An early event in
the damage cascade is angiotensin II upregulation, which stimulates
tumor necrosis factor- (TNF-
) production and TGF-
expression
(5, 14, 29, 30,
33). These cytokines activate nuclear factor
B
(NF-
B) (22, 34), a crucial transcription
factor in fibroblasts, macrophages, and epithelial cells, leading to
expression of
-SMA, type IV collagen, osteopontin, MCP-1,
intercellular adhesion molecule 1, and vascular cell adhesion molecule
1, in addition to other genes (44), involved in renal
cellular transformation and apoptosis as well as interstitial inflammation and subsequent fibrosis. The damage cascade stimulated by
UUO closely resembles that produced by several forms of renal injury
(5, 25, 28, 53).
Suppression of this damage cascade might prevent fibrogenesis and
preserve renal function. In fact, we and others have demonstrated that
ACE inhibition, type 1 angiotensin II receptor blockade, or reduction
of angiotensin gene expression is protective against the activation of
the damage cascade and against interstitial fibrosis (10,
18, 23, 24, 31).
As shown here, UUO was associated with a major remodeling of the outer and inner medullary tubular epithelium, which by 5 days lost cell-cell and cell-matrix contact and became effaced and flattened due in part to increased epithelial cell apoptosis. This pattern of tubular cell damage was greatest in the medulla, as has been reported in the early stages of UUO (8, 9), leading to tubular atrophy, which is a prominent component of the renal injury after obstruction.
OP-1 administration dose dependently inhibited epithelial cell apoptosis and thereby preserved tubular epithelial morphology, contributing to prevention of tubular atrophy. UUO is known to stimulate phenotypic transformation of tubular cells that acquire mesenchymal characteristics (7, 8, 26) and have increased rates of apoptosis (8, 26). OP-1 suppressed UUO- stimulated loss of tubular epithelium due to apoptosis and prevented the transformation of renal cells into interstitial myofibroblasts. This suggests that, whereas OP-1 prevented tubular cell apoptosis as previously reported (50), it further appears to have maintained the phenotype of tubular cells and the interstitial fibroblasts. Both tubular cells and interstitial fibroblasts are subjected to phenotypic alterations as a result of UUO (8, 18, 26, 42, 43). The preponderance of evidence is that phenotypic alteration of epithelial and fibroblastic cells to myofibroblasts is detrimental and leads to a progressive loss of renal function (18, 23, 24, 42, 43).
As shown by the present study, the tubular epithelial injury produced by UUO was associated with a loss of OP-1 expression in the obstructed kidney. The pattern of OP-1 expression in the medulla became limited to the nuclear membrane of the cells in the otherwise atrophic epithelia. Nuclear localization of a renal growth factor or of a morphogen after injury appears not to be unique to OP-1. Recently, fibroblast growth factor-2 has been found in cytoplasmic and nuclear locations within epithelial cells of the human kidney (19). OP-1 location in the nucleus may preserve renal epithelial cells by maintaining the differentiation program of the cell in the face of injury and pressures to dedifferentiate or progress into apoptosis. Our immunohistochemical localization of OP-1 in the collecting ducts of normal kidneys was compatible with prior studies (47, 50) that included message localization by in situ hybridization. The loss of OP-1 expression during UUO, especially in the medulla, was compatible with studies of renal OP-1 expression after ischemic injury (1, 50).
In comparison to OP-1, enalapril treatment also inhibited interstitial
fibrosis stimulated by UUO, but in agreement with Fern et al.
(18), it failed to prevent the tubular cell atrophy and to
protect the expression of OP-1. The magnitude of the protective actions
of enalapril in the present study was similar to our previous reports
(23, 33). In recent years, the discovery of
the protective actions of ACE inhibition against the progressive
destruction of kidney parenchyma by several diseases has begun a new
era of kidney failure prevention (3, 35,
54). ACE inhibition is protective against the renal
failure produced in several animal models of kidney injury including
UUO (3, 4, 54). The effects of
ACE inhibition are mediated, in part, through inhibition of intrarenal
paracrine functions of angiotensin II, which activates a damage cascade
of cytokines and transcription factors in response to renal injury.
OP-1 also inhibited the activation of the damage cascade as part of its
mechanism of renal protection (Chaudhary, Morrissey, and Hruska,
unpublished observations); epithelial integrity or direct inhibition of
cytokine production remains to be determined, and the results will be
reported separately. OP-1 was similar to but greater than enalapril in
its protective action against tubulointerstitial fibrosis. In addition,
OP-1 preserved tubular epithelial structure and prevented tubular
atrophy. ACE inhibition decreases the activity of the damage cascade by
suppressing UUO stimulation of TGF-, TNF-
, and NF-kB, which are
mediated by angiotensin II (32, 34).
Approximately 50% of the stimulation of this damage cascade, after
UUO, is due to angiotensin II (18), and our data suggest
that a higher percentage, >50%, is suppressed by OP-1. Thus OP-1 may
function as a renal homeostasis signal by providing a survival signal
to epithelial cells, protecting tubular epithelial cell phenotype, and
suppressing gene activation associated with injury.
A second mechanism by which ACE inhibition is renoprotective derives from its actions to preserve RBF. UUO stimulates angiotensin II-mediated vasoconstriction, which ACE inhibition prevents. In our studies, the effect of ACE inhibition on RBF was not apparent because none of the ACE-treated rats had a return of urine flow in the postobstructed kidney, and PAH clearances were not possible. OP-1, on the other hand, was 50% effective at restoring urine flow after 5 days of obstruction before release of UUO and 5 days of recovery. RBF in the postobstructed kidneys estimated by PAH clearances was ~60% of the sham-operated single-kidney RBF, which was >30% retention of GFR. This indicates that OP-1 was also protective of RBF, and that this was a second mechanism of action that must be considered in the renoprotective actions of OP-1, similar to the actions of enalapril. By analysis of RBF in the contralateral kidney after UUO, no significant differences were found between sham-operated and vehicle-treated or OP-1-treated kidneys. However, enalapril significantly increased RBF in the contralateral kidney. This appeared to be a direct action of enalapril because the degree of compensatory hypertrophy of the contralateral kidney would have been expected to be greatest in the vehicle-treated group of rats.
In summary, we show that OP-1 administration is protective against the tubulointerstitial fibrosis and renal destruction produced by UUO. The mechanism of action was, in part, inhibition of renal tubular cell apoptosis, which lead to preservation of tubular epithelial integrity, as well as epithelial cell phenotype, and inhibition of interstitial infiltration and interstitial fibroblast transformation. Secondary actions on preservation of renal blood were also contributory. The question of whether OP-1 administration at the time of relief of obstruction will also be therapeutic awaits further study. Our results clearly suggest that OP-1 is an important renal homeostatic factor with actions to suppress the impact of renal injury.
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ACKNOWLEDGEMENTS |
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The authors thank Daniel Martin and Sue King for the technical assistance with the renal function measurements and Ruth McCracken for the immunohistochemistry determinations.
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FOOTNOTES |
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These studies were supported by National Institutes of Health Grants P01-DK-09976 (S. Klahr, J. Morrissey and K. A. Hruska), AR-39561 (K. A. Hruska), and AR-32087 (K. A. Hruska) and by a grant from Creative Biomolecules, Hopkinton, MA.
Address for reprint requests and other correspondence: K. A. Hruska, Barnes-Jewish Hospital North, Mailstop #90-32-648, 216 S Kings-highway, St. Louis, MO 63110 (E-mail: khruska{at}imgate.wustl.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 18 January 2000; accepted in final form 7 April 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Almanzar, MM,
Frazier KS,
Dube PH,
Piqueras AN,
Jones WK,
Charette MF,
and
Paredes AL.
Osteogenic protein-1 mRNA expression is selectively modulated after acute ischemic renal injury.
J Am Soc Nephrol
9:
1456-1463,
1998[Abstract].
2.
Alvarez, RJ,
Sun MJ,
Haverty TP,
Iozzo RV,
Myers JC,
and
Neilson EG.
Biosynthetic and proliferative characteristics of tubulointerstitial fibroblasts probed with paracrine cytokines.
Kidney Int
41:
14-23,
1992[ISI][Medline].
3.
Anderson, S,
Meyer TW,
Rennke HG,
and
Brenner BM.
Control of glomerular hypertension limits glomerular injury in rats with reduced renal mass.
J Clin Invest
76:
612-619,
1986[ISI].
4.
Bidani, AK,
and
Griffin KA.
Experimental models of renal mass reduction and their relevance to clinical medicine.
Kidney Curr Surv World Lit
4:
4-9,
1995.
5.
Border, WA,
and
Noble NA.
Interactions of transforming growth factor-beta and angiotensin II in renal fibrosis.
Hypertension
31:
181-188,
1998
6.
Bruzzi, I,
Benigni A,
and
Ramuzzi G.
Role of increased glomerular protein traffic in the progression of renal failure.
Kidney Int
62:
S29-S31,
1997.
7.
Chevalier, RL,
Goyal S,
Wolstenholme JT,
and
Thornhill BA.
Obstructive nephropathy in the neonatal rat is attenuated by epidermal growth factor.
Kidney Int
54:
38-47,
1998[ISI][Medline].
8.
Chevalier, RL,
Kim A,
Thornhill BA,
and
Wolstenholme JT.
Recovery following relief of unilateral ureteral obstruction in the neonatal rat.
Kidney Int
55:
793-807,
1999[ISI][Medline].
9.
Chevalier, RL,
Thornhill BA,
and
Wolstenholme JTKA
Unilateral ureteral obstruction in early development alters renal growth: dependence on the duration of obstruction.
J Urol
161:
309-313,
1999[ISI][Medline].
10.
Chung, KH,
Gomez RA,
and
Chevalier RL.
Regulation of renal growth factors and clusterin by angiotensin AT1 receptors during neonatal ureteral obstruction.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F1117-F1123,
1995
11.
Couser, WG.
Mediators of immune glomerular injury.
Clin Invest
71:
8-11,
1993.
12.
Couser, WG.
Pathogenesis of glomerulonephritis.
Kidney Int
44:
S519-S526,
1993.
13.
Diamond, JR,
Kees-Folts D,
Ding G,
Frye JE,
and
Restrepo NC.
Macrophages, monocyte peptide-1, and TGF-1 in experimental hydronephrosis.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F926-F933,
1994
14.
Douglas, JG,
Romero M,
and
Hopper U.
Signaling mechanisms coupled to the angiotensin receptor of proximal tubule epithelium.
Kidney Int
38:
S43-S47,
1990[ISI].
15.
Dudley, AT,
Lyons KM,
and
Robertson EJ.
A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye.
Genes Dev
9:
2795-2807,
1995[Abstract].
16.
Eddy, AA.
Interstitial inflammation and fibrosis in rats with diet-induced hypercholesterolemia.
Kidney Int
50:
1139-1149,
1996[ISI][Medline].
17.
El-Dahr, SS,
Gee J,
Dipp S,
Hanss BG,
Vari RC,
and
Chao J.
Upregulation of renin-angiotensin system and downregulation of kallikrein in obstructive nephropathy.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F874-F881,
1993
18.
Fern, RJ,
Yesko CM,
Thornhill BA,
Kim H-Y,
Smithies O,
and
Chevalier RL.
Reduced angiotensinogen expression attenuates renal interstitial fibrosis in obstructive nephropathy in mice.
J Clin Invest
103:
39-46,
1999
19.
Floege, J,
Hudkins KL,
Eitner F,
Cui Y,
Morrison RS,
Schelling MA,
and
Alpers CE.
Localization of fibroblast growth factor-2 (basic FGF) and FGF receptor-1 in adult human kidney.
Kidney Int
56:
883-897,
1999[ISI][Medline].
20.
Gröne, EF,
Walli AK,
Gröne HJ,
Miller B,
and
Seidel D.
The role of lipids in nephrosclerosis and glomerulosclerosis.
Atherosclerosis
107:
1-13,
1994[ISI][Medline].
21.
Helder, MN,
Ozkaynak E,
Sampath KT,
Lluyten FP,
Latin V,
Oppermann H,
and
Vukicevic S.
Expression pattern of osteogenic protein-1 (bone morphogenetic protein-7) in human and mouse development.
J Histochem Cytochem
43:
1035-1044,
1995
22.
Ibrahim, HN,
Rosenberg ME,
and
Hostetter TH.
Role of the renin-angiotensin-aldosterone system in the progression of renal disease: a critical review.
Semin Nephrol
17:
431-440,
1997[ISI][Medline].
23.
Ishidoya, S,
Morrissey J,
McCracken R,
and
Klahr S.
Delayed treatment with enalapril halts tubulointerstitial fibrosis in rats.
Kidney Int
49:
1110-1119,
1996[ISI][Medline].
24.
Ishidoya, S,
Morrissey J,
McCracken R,
Reyes A,
and
Klahr S.
Angiotensin II receptor antagonist ameliorates renal tubulointerstitial fibrosis caused by unilateral ureteral ligation.
Kidney Int
47:
1285-1294,
1995[ISI][Medline].
25.
Johnson, RJ,
Alpers CE,
Yoshimura A,
Lombardi D,
Pritzl P,
Floege J,
and
Schwartz SM.
Renal injury from angiotensin II-mediated hypertension.
Hypertension
19:
464-467,
1992[Abstract].
26.
Johnson, RJ,
Hugo C,
Haseley C,
Pichler RH,
Bassuk J,
Thomas S,
Suga S,
Couser WG,
and
Shankland SJ.
Mechanisms of progressive glomerulosclerosis and tubulointerstitial fibrosis.
Clin Exp Nephrol
2:
307-312,
1998.
27.
Jones, WK,
Richmond EA,
White K,
Sasak HKW,
Smart J,
Oppermann H,
Rueger DC,
and
Tucker RF.
Osteogenic protein-1 (OP-1) expression and processing in Chinese hamster ovary cells: isolation of a soluble complex containing the mature and pro-domains of OP-1.
Growth Factors
11:
215-225,
1994[ISI][Medline].
28.
Kagami, S,
Border WA,
Miller DE,
and
Noble NA.
Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor-beta expression in rat glomerular mesangial cells.
J Clin Invest
93:
2431-2437,
1994[ISI][Medline].
29.
Kaneto, H,
Morrissey J,
and
Klahr S.
Increased expression of TGF-1 mRNA in the obstructed kidney of rats with unilateral ligation.
Kidney Int
44:
313-321,
1993[ISI][Medline].
30.
Kaneto, H,
Morrissey J,
McCracken R,
Ishidoya S,
Reyes A,
and
Klahr S.
The expression of mRNA for tumor necrosis factor increases in the obstructed kidney of rats soon after unilateral ureteral ligation.
Nephrology
2:
161-166,
1996[ISI].
31.
Kaneto, H,
Morrissey J,
McCracken R,
Reyes A,
and
Klahr S.
Enalapril reduces collagen type IV synthesis and expansion on the interstitium in the obstructed kidney.
Kidney Int
45:
1637-1647,
1994[ISI][Medline].
32.
Klahr, S. S.
Nephrology forum: obstructive nephropathy.
Kidney Int.
54:
286-300,
1998[ISI][Medline].
33.
Klahr, S,
Ishidoya S,
and
Morrissey J.
Role of angiotensin II in the tubulointerstitial fibrosis of obstructive nephropathy.
Am J Kidney Dis
26:
141-146,
1995[ISI][Medline].
34.
Klahr, S,
and
Morrissey J.
Angiotensin II and gene expression in the kidney.
Am J Kidney Dis
31:
171-176,
1998[ISI][Medline].
35.
Lewis, E J,
Hunsicker LG,
Bain RP,
and
Rohde RD.
The effect of angiotensin-converting-enzyme inhibition on diabetic nephropathy.
N Engl J Med
329:
1456-1462,
1993
36.
Luo, G,
Hofmann C,
Bronckers AL,
Sohocki M,
Bradley A,
and
Karsenty G.
BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning.
Genes Dev
9:
2808-2820,
1995[Abstract].
37.
Massaque, J.
The transforming growth factor- family.
Annu Rev Cell Biol
6:
597-641,
1990[ISI].
38.
Miller, SB,
Martin DR,
Kissane J,
and
Hammerman MR.
Hepatocyte growth factor accelerates recovery from acute ischemic renal injury in rats.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F129-F134,
1994
39.
Miller, SB,
Moulton M,
O'Shea M,
and
Hammerman MR.
Effects of IGF-I on renal function in end-stage renal failure.
Kidney Int
46:
201-207,
1994[ISI][Medline].
40.
Modi, KS,
Morrissey J,
Shah SV,
Schreiner GF,
and
Klahr S.
Effects of probucol on renal function in rats with bilateral ureteral obstruction.
Kidney Int
38:
843-850,
1990[ISI][Medline].
41.
Moller, JC,
Skriver E,
Olsen S,
and
Maunsbach AB.
Ultrastructural analysis of human proximal tubules and cortical interstitium in chronic renal disease (hydronephrosis).
Virchows Arch
402:
209-237,
1984.
42.
Nagle, RB,
Johnson ME,
and
Jervis HR.
Proliferation of renal interstitial cells following injury induced by ureteral obstruction (Abstract).
Lab Invest
35:
18,
1976[ISI][Medline].
43.
Ng, YY,
Huang TP,
Yang WC,
Chen ZP,
Yang AH,
Mu W,
Nikolic-Paterson DJ,
and
Atkins RCLHY
Tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats.
Kidney Int
54:
864-876,
1998[ISI][Medline].
44.
Oikawa, T,
Freeman M,
Lo W,
Vaughan DE,
and
Fogo A.
Modulation of plasminogen activator inhibitor-1 in vivo: a new mechanism for the anti-fibrotic effect of renin-angiotensin inhibition.
Kidney Int
51:
164-172,
1997[ISI][Medline].
45.
Ozkaynak, E,
Schnegelsberg PN,
and
Oppermann H.
Murine osteogenic protein 1 (OP-1): high levels of mRNA in kidney.
Biochem Biophys Res Commun
179:
116-123,
1991[ISI][Medline].
46.
Sampath, TK,
Maliakal JC,
Hauschka PV,
Jones WK,
Sasak H,
Tucker RF,
White KH,
Coughlin JE,
Tucker MM,
Pang RHL,
Corbett C,
Ozkaynak E,
Oppermann H,
and
Rueger DC.
Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro.
J Biol Chem
267:
20352-20362,
1992
47.
Simon, M,
Maresh JG,
Harris SE,
Hernandez JD,
Arar M,
Olson MS,
and
Abboud HE.
Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney.
Am J Physiol Renal Physiol
276:
F382-F389,
1999
48.
Solursh, M,
Langille RM,
Wood J,
and
Sampath TK.
Osteogenic protein-1 is required for mammalian eye development.
Biochem Biophys Res Commun
218:
438-443,
1996[ISI][Medline].
49.
Truong, LD,
Sheikh-Hamad D,
Chakraborty S,
and
Suki WN.
Cell apoptosis and proliferation in obstructive uropathy.
Semin Nephrol
18:
641-651,
1998[ISI][Medline].
50.
Vukicevic, S,
Basic V,
Rogic D,
Basic N,
Shih M-S,
Shepard A,
Jin D,
Dattatrenamurty B,
Jones W,
Dorai H,
Ryan S,
Griffiths D,
Maliakal J,
Jelic M,
Pastorcic M,
Stavlijenic A,
and
Sampath TK.
Osteogenic protein-1 (bone morphogenetic protein-7) reduces severity of injury after ischemic acute renal failure in rat.
J Clin Invest
102:
202-214,
1998
51.
Vukicevic, S,
Kopp JB,
Luyten FP,
and
Sampath TK.
Induction of nephrogenic mesenchyme by osteogenic protein 1 (bone morphogenetic protein 7).
PNAS
93:
9021-9026,
1996
52.
Yoo, KH,
Norwood VF,
El-Dahr SS,
Yosipiv I,
and
Chevalier RL.
Regulation of angiotensin II AT1 and AT2 receptors in neonatal ureteral obstruction.
Am J Physiol Regulatory Integrative Comp Physiol
273:
R503-R509,
1997
53.
Yoo, KH,
Thornhill BA,
Wolstenholme JT,
and
Chevalier RL.
Tissue-specific regulation of growth factors and clusterin by angiotensin II.
Am J Hypertens
11:
715-722,
1998[ISI][Medline].
54.
Zatz, R,
Dunn BR,
Meyer TW,
Anderson S,
Rennke HG,
and
Brenner BM.
Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension.
J Clin Invest
77:
1925-1930,
1986[ISI][Medline].