Osteogenic protein-1 prevents renal fibrogenesis associated with ureteral obstruction

Keith A. Hruska1,2, Guangjie Guo1,2, Magdalena Wozniak1, Daniel Martin1, Steven Miller1, Helen Liapis3, Kenneth Loveday4, Saulo Klahr1, T. Kuber Sampath4, and Jeremiah Morrissey1,2

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BONE MORPHOGENETIC PROTEIN-7 (BMP-7), also known as osteogenic protein-1 (OP-1), a member of the transforming growth factor-beta (TGF-beta ) 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-beta 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 wt-1 · 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).

The effects of OP-1 were compared with placebo (vehicle-treated) and to a positive control (enalapril treatment) known to ameliorate the fibrogenesis resulting from UUO in previous studies in our laboratory and other laboratories (23, 31). The enalapril dose is that used in our previous studies in which its effectiveness was demonstrated (23, 31). It is a high dose, much greater than that required for modulation of the systemic renin-angiotensin system. Studies were performed in a blinded fashion. Treatment solutions were made by an investigator who was not part of the study and were assigned a letter. Investigators were unaware of the content of the various treatments until after clearance studies and histopathological grading were completed. The investigators (Morrissey J and Liapis H) grading the histopathological slides were also blinded to the treatment groups.

Renal function was determined by measuring glomerular filtration rate (GFR) of individual kidneys by inulin clearances and by estimating renal blood flow (RBF) by PAH clearances as previously reported (38, 39). Rats were anesthetized with intraperitoneal Nembutal, and catheters were placed in the femoral artery and vein and both ureters. The animals were allowed to awaken in a Plexiglas restrainer, and the clearance studies were performed in awake animals.

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 alpha -smooth muscle actin (alpha -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 and alpha -SMA expression. Primary antibodies to collagen type IV (goat polyclonal) from Southern Biotechnology (Birmingham, AL) and antibodies to alpha -SMA (clone HHF35) were obtained from BioGenex Laboratories (San Ramon, CA). We have previously reported the use of these antibodies to assess fibrosis (23, 24, 31). The location of the primary antibodies was visualized by using horseradish peroxidase or alkaline phosphatase-linked second antibodies as previously reported.

Quantitation 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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Fig. 1.   Coronal sections of kidneys stained with the Masson's Trichrome stain for collagen. With this stain, collagen fibrils stain blue, whereas the cells stain red. A and B are sections from the cortices of both kidneys of sham-operated animals. Collagen is detected in the renal capsule, around larger vessels, and in Bowman's capsule. Little collagen is detectable in the renal interstitium (C and D) sections from two different areas of the obstructed kidney of a vehicle-treated animal. There are widespread and diffuse blue stain of the interstitial collagen and a major interstitial cellular infiltrate. E and F: 2 sections of the cortex of an obstructed kidney from an animal treated with 100 µg/day of osteogenic protein-1 (OP-1). The amount of interstitial collagen is only slightly more than that of the sham-operated kidneys. G and H: 2 sections from the cortex of a kidney from an animal treated with 300 µg/day of OP-1. The amount of interstitial collagen is indistinguishable from the interstitial collagen deposition in the sham-operated animals.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2.   A and B: sections from 2 areas of the cortex of the contralateral kidney of a vehicle-treated animal. The collagen staining in the contralateral kidneys of all the groups did not differ significantly from that of the kidneys of the sham-operated animals.



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Fig. 3.   Interstitial volume (Vint) after release of unilateral ureteral obstruction (UUO) at day 5 and analysis at day 10. Interstitial volume was increased to 474 ± 47% in obstructed kidneys from 97 ± 0.2% in sham-operated control kidneys. All prevention regimens significantly reduced the increase in interstitial volume after UUO. OP-1 was significantly more effective than enalapril, and 300 µg/kg of soluble OP-1 (sOP-1) every other day were more effective than 100 µg/kg of the same treatement.

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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Fig. 4.   Interstitial type IV collagen deposition after release of UUO at day 5 and analysis at day 10. A and B: 2 sham-operated kidney section and contralateral kidney sections, respectively. The perceptible interstitial volume is small, and the basement membranes of adjacent tubules and peritubular capillaries are essentially abutting each other. C and D: in the cortex of ureteral obstructed kidneys from vehicle-treated animals, type IV collagen staining in the expanded interstitium is easily seen in the space between adjacent tubules. In addition, the basement membranes of engorged peritubular capillaries are easily observed. E and F: sections from obstructed kidneys of 2 animals treated with 100 µg/kg of sOP-1. G and H: sections from obstructed kidneys of 2 animals treated with 300 µ/kg of sOP-1. There was a marked diminution in the accumulation of type IV collagen in the interstitium of the kidneys of the animals treated with 100 or 300 µg/kg of OP-1. The reduction in interstitial type IV collagen was nearly back to baseline and it is fairly close to the normal-appearing interstitium of the sham-operated animals.



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Fig. 5.   Analysis of type IV collagen deposition in the interstitium. The scoring of type IV collagen deposition revealed significant reductions in the accumulation of collagen IV stimulated by UUO induced by all of the treatment groups. The effect of OP-1 was significantly greater than the protection afforded by the enalapril treatment.

We next examined the expression of alpha -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, alpha -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 alpha -SMA (Fig. 6D), as demonstrated by a lesser histological scoring of alpha -SMA (Fig. 6E). The effect of OP-1 on alpha -SMA histological scores was dose dependent, and the 300 µg/kg dose was significantly more effective than enalapril in preventing accumulation of cells expressing alpha -SMA in the interstitium.


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Fig. 6.   Interstitial alpha -smooth muscle actin expression after release of UUO at day 5 and analysis at day 10. A and B: in sham-operated animals and contralateral kidneys of animals with UUO, respectively, alpha -smooth muscle actin was detected only in the vascular wall of vessels. A significant background stain is detected by using the rhodamine-labeled second antibody. C: marked expression of alpha -smooth muscle actin in the expanded interstitium of a cortical section of a ureteral obstructed kidney from vehicle-treated animal. D: as shown by a cortical section of a ureteral obstructed kidney from an animal treated with 100 µg/day of OP-1, alpha -smooth muscle actin expression in the interstitium is markedly reduced compared with the kidneys of the vehicle-treated animals. The alpha -smooth muscle actin expression in the contralateral kidneys of both the 100 and 300 µg/day of OP-1 treatment revealed alpha -smooth muscle actin levels similar to the contralateral kidneys or those from sham-operated animals. E: the alpha -smooth muscle actin score observed in sections from vehicle-treated animals was significantly reduced by OP-1 and enalapril therapy. The 300 µg/kg dose of OP-1 was significantly more protective than either the 100 µg/kg dose or enalapril. NS, not significant.

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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Fig. 7.   Interstitial cellular infiltrate following release of UUO at day 5 and analysis at day 10 Macrophage infiltration detected by reactivity to the anti-ED-1 antibody demonstrated a reduction in interstitial ED-1 positive cells from 90 to 42 and 31×200 field in the 100 and 300 µg/kg sOP-1 treatment groups, respectively. OP-1 (300 µg/kg) was significantly more protective than enalapril treatment.

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 · min-1 · 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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Fig. 8.   Renal function after release of UUO at day 5 and clearance studies performed on day 10. A: urine flow in the postobstructed kidneys was restored in 50% of the OP-1 animals but in none of the vehicle- or enalapril-treated animals. Urine flow was present in 100% of the contralateral kidneys of all the groups. B: inulin clearances in the sham-operated and sOP-1- treated animals with urine flow. In the absence of urine flow, clearance studies could not be performed in the other animals, and they were assigned a clearance of 0. C: p-aminohippurate (PAH) clearances in the sham-operated and the sOP-1-treated animals with urine flow. In the absence of urine flow, clearance studies could not be performed in the other animals and they were assigned a clearance of 0.

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 alpha -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 alpha -SMA (Fig. 9, A-C). Visual inspection of the immunocytochemistry for collagen IV and alpha -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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Fig. 9.   Histological parameters of the renal interstitium of rats with UUO sustained for 10 days. A: Vint. B: type IV collagen accumulation. C: alpha -smooth muscle actin expression. D: macrophage infiltration in kidneys from rats with sustained UUO for 10 days.

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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Fig. 10.   OP-1 prevented tubular atrophy. Coronal sections of kidneys were stained with the periodic acid Schiff stain to highlight basement membranes. A: sham-operated kidney basement membranes are thinned, smooth, and there is no interstitial infiltrate. B: vehicle-treated kidney UUO for 5 days with release and analysis at day 10. Heavy interstitial infiltrate and small atrophic tubules with thickened basement membranes were widely observed. Some tubules have white blood cells in the lumens. C: 100 µg/kg sOP-1 every other day. The interstitial infiltrate is markedly diminished, but an occasional basement membrane continues to demonstrate thickening, but the atrophy has been largely prevented. D: 300 µg/kg of sOP-1 every other day. The normal pattern of basement membrane expression has basically been preserved. E: enalapril, 25 mg/kg daily. The cellular infiltrate is slightly less than in B, but the tubular atrophy is essentially unchanged. F: quantitation of atrophic tubules derived from fifty ×40 fields/kidney section. Both of the sOP-1 dosages (n = 6/group) were significantly improved compared with vehicle (n = 7) or the enalapril-treated group (n = 4).

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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Fig. 11.   OP-1 inhibited UUO-stimulated apoptosis. Coronal sections of kidneys were stained by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptotic nuclei. A: sham-operated kidney (n = 4 rats). B: vehicle-treated obstructed kidney. C: 100 µg/kg sOP-1 every other day (n = 6 rats). D: 300 µg/kg sOP-1 every other day (n = 6 rats). E: enalapril 25 mg/kg daily (n = 4 rats). F: quantitation of TUNEL-positive nuclei.

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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Fig. 12.   Renal OP-1 expression in the inner medulla after ureteral obstruction. The medullary tubular epithelial cells of sham-operated animals (A) express BMP-7 except for occasional cells. After release of UUO at day 5 and analysis at day 10, the no. of cells strongly expressing bone morphogenetic protein-7 (BMP-7) was markedly reduced in the medullary tubular epithelia (B), and there was an apparent shift in OP-1 expression from a diffuse cytoplasmic location to the nucleus of prominent rounded cells. OP-1 (100 µg/kg) partially restored the pattern of OP-1 expression (C), and OP-1 (300 µg/kg) normalized the expression of OP-1 (D). Enalapril treatment did not protect OP-1 expression in the inner medulla (E). The negative controls for the immunostaining (F, inner medulla) using an irrelevant IgG were all essentially black.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (TNF-alpha ) production and TGF-beta expression (5, 14, 29, 30, 33). These cytokines activate nuclear factor kappa B (NF-kappa B) (22, 34), a crucial transcription factor in fibroblasts, macrophages, and epithelial cells, leading to expression of alpha -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-beta , TNF-alpha , 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.


    ACKNOWLEDGEMENTS

The authors thank Daniel Martin and Sue King for the technical assistance with the renal function measurements and Ruth McCracken for the immunohistochemistry determinations.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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