Bone Morphogenic Protein7 Inhibits Progression of Chronic Renal Fibrosis Associated with two Genetic Mouse Models

Bone Morphogenic Protein-7 Inhibits Progression ofChronic Renal Fibrosis Associated with two GeneticMouse Models

Michael Zeisberg, Cindy Bottiglio, Navin Kumar, Yohei Maeshima, Frank Strutz*, Gerhard A.

Müller* and Raghu Kalluri

Center for Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center and

Harvard Medical School, Boston, MA 02215, USA, *Department of Nephrology and

Rheumatology, Georg-August University Medical Center, Robert-Koch-Str. 40, 37075

Göttingen, Germany

Running title: BMP 7 inhibits renal fibrosis

Correspondence should be addressed to:Dr. Raghu Kalluri

Associate Professor of Medicine

Harvard Medical School

Director, Center for Matrix Biology

Department of Medicine, Dana 514

Beth Israel Deaconess Medical Center

330 Brookline Avenue

Boston, MA 02215Tel#617-667-0445Fax#617-667-2562e-Mail:[email protected]

Copyright (c) 2003 by the American Physiological Society.

Articles in PresS. Am J Physiol Renal Physiol (August 12, 2003). 10.1152/ajprenal.00191.2002

American Journal of Physiology � Renal Physiology F-00191-2002 BMP-7 inhibits renal fibrosisZeisberg/Bottiglio/Kumar/Maeshima/Strutz/Muller/Kalluri

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Abstract

Tubulointerstitial fibrosis is a hallmark feature of chronic renal injury. Specific therapies to

control the progression of renal fibrosis towards end-stage renal failure are still limited. Previous

studies have demonstrated that expression of endogenous BMP-7 is reduced in the kidneys of

several inducible mouse models of acute and chronic renal disease, and that administration of

exogenous recombinant human bone morphogenic protein-7 (rhBMP-7) has a beneficial effect

on kidney function. Here we report that treatment with rhBMP-7 leads to improved renal

function, histology and survival in a3(IV) collagen deficient mice and in MRL/MpJlpr/lpr lupus mice,

two different genetic models for chronic renal injury and fibrosis. Such therapeutic benefit is

also associated with a significant decrease in the expression of pro-fibrotic molecules, such as

collagen I and fibronectin, in renal fibroblasts. Additionally, rhBMP-7 induces the expression of

active matrix metalloproteinase-2 (MMP-2), which is potentially important for the removal of

fibrotic matrix. Collectively these studies provide further evidence for rhBMP-7 as an important

bone protein with protective function against renal pathology.

Keywords: Bone Morphogenic Protein-7, Tubulointerstitial Fibrosis, Fibroblasts, OsteogenicProtein-1, Matrix Metalloproteinases


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Despite significant advances in understanding the mechanistic pathways mediating the

progression of chronic renal disease towards end-stage renal failure (ESRF), options for

effective pharmacotherapy are still very limited (7, 18, 25, 37). In progressive chronic renal

disease, a self-preserving mechanism dominates independent of the underlying disease, further

validating the notion for existence of a potential common pathway leading to ESRF (13, 39).

Progressive renal disease is almost invariantly characterized by the triad of glomerular injury,

tubulointerstitial fibrosis and tubular atrophy (4, 16).

Bone Morphogenic Protein-7 (syn. Osteogenic Protein-1/ OP-1) is a member of the TGF-

b superfamily that plays a crucial role in renal development (12, 28, 34). Expression of BMP-7

correlates with condensation of the metanephric mesenchyme to generate epithelium, which

eventually leads to formation of tubules and glomeruli (28, 36, 40, 50, 51). In the adult mouse

kidney, BMP-7 is expressed in the collecting duct, thick ascending limb, distal convoluted

tubule, and podocytes within glomeruli (17). Acute renal injury associated with tubular necrosis

leads to reduction in the expression of tubular BMP-7, and after recovery of tubular and

glomerular damage BMP-7 expression is restored (42, 49). Administration of exogenous

recombinant human BMP-7 (rhBMP-7) restores normal tubule architecture in rat models of

ischemic acute renal injury and it also restores tubular homeostasis in a mouse model of

nephrotoxic serum nephritis (42, 49, 56). Furthermore, administration of exogenous rhBMP-7

inhibits progression of chronic renal disease in a rat model of unilateral urethral obstruction

(UUO) and a rat model of streptozotocin (STZ) induced diabetic nephropathy (21, 24). Here we

report that rhBMP-7 exhibits inhibitory effects on chronic renal disease and fibrosis in

MRL/MpJlpr/lpr lupus mice and in a3(IV) collagen deficient (Col4A3-/-) mice. We further report that

rhBMP-7 exerts anti-fibrogenic effects on renal fibroblasts, both in the in vitro and in vivo

settings. We also demonstrate that treatment of mice with rhBMP-7 over a four-month period


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does not induce significant side effects and is non-toxic. Collectively, these results suggest that

rhBMP-7 is a potential candidate for the treatment of chronic renal failure.

Methods

Materials. For in vivo studies, recombinant human BMP-7 homodimer, non-covalently

attached to prodomain protein (referred to as soluble rhBMP-7) was used (Curis, Inc.,

Cambridge, MA) (21). In cell culture experiments, rhBMP-7, dissolved in 24 mM Na acetate [pH

4.5] containing 1% mannitol, was used (Curis, Inc. Cambridge, MA) (17). Details regarding

these recombinant proteins have been published previously (17, 21).

Mice: Female normal mice on an MRL/MpJ background and autoimmune disease-prone

MRL/MpJlpr/lpr mice were purchased from the Jackson Laboratories (Bar Harbor, ME) and

maintained in the BIDMC animal facility (11). Col4A3-/- mice on a mixed background of C57BL/6

and 129/Sv were maintained at the BIDMC animal facility. Homozygous deletion of Col4A3-/-

was confirmed by PCR as described previously (30). The Col4A3-/- mice were either obtained

from Jackson Laboratories (Bar Harbor, ME) or were kindly provided by Dr. Jeffrey Miner (St.

Louis, MO). Mice were fed standard mouse chow and water ad libitum. All mouse studies

followed the standards approved by the institutional animal care committee.

Experimental design. Normal mice on an MRL/MpJ background (n=24) were treated

with vehicle buffer alone or received rhBMP-7 at 30 mg/kg, 100 mg/kg and 300 mg/kg for 16

weeks to assess long term toxicology of rhBMP-7. MRL/MpJlpr/lpr mice (lupus nephritis mice)

were treated for 12 weeks, starting treatment at the age of four weeks and the study was

terminated at the age of 16 weeks. Mice were divided into a control group (n=6 each) that


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received vehicle buffer alone and three experimental groups (n=6 each) that received rhBMP-7

at 30 mg/kg, 100 mg/kg and 300 mg/kg. Due to the different kinetics of disease progression,

COL4A3-/- mice were treated for 8 weeks and the study was terminated at the age of 14 weeks.

Col4A3-/- mice (n=14) were divided into a control group (n=7) that received vehicle buffer alone

and a treatment group that received 300 mg/kg rhBMP-7. rhBMP-7 was administered

intraperitoneally (i.p.) three times a week. Serum was obtained every two weeks from baseline

to death on all mice. Urine was collected every two weeks in metabolic cages.

Histologic Assessment of renal injury. Tissue from kidneys, liver, heart, spleen and

brain was fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with

hematoxylin and eosin (HE), Masson�s Trichrome Stain (MTC), as well as with periodic acid-

Schiffs stain (PAS). Extent of renal injury was estimated by morphometric assessment of the

tubulointerstitial injury and glomerular damage. The relative interstitial volume was evaluated by

morphometric analysis using a 10 mm2 graticule fitted into the microscope. Five randomly

selected cortical areas, which included glomeruli, were evaluated for each animal (5, 45).

Tubules were evaluated for their widened lumen and thickened basement membranes to

estimate percentage of atrophic tubules (21). 100 glomeruli in each kidney were evaluated for

crescent formation, the crescentic index reflects the percentage of crescentic glomeruli(47).

Glomerulosclerosis was scored for severity (0-4, 0=normal, 4=maximum severity) as described

by Raij (38). Nuclei in 50 glomeruli of similar size in each kidney were counted to estimate

glomerular cellularity (29).

Immunohistochemistry. Kidney tissue samples were frozen in liquid nitrogen and

processed by indirect immunofluorescence technique as previously described (45). Frozen


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sections were stained with the primary antibody, raised against type I collagen (Southern

Biotechnology Associates Inc., Birmingham, AL), MMP-2 (Chemicon, Temecula, CA), MMP-9

(Chemicon) or BMP-7 (Curis, Inc.). After washing with Tris-buffered saline, sections were

incubated with FITC- and Rhodamine-conjugated secondary antibodies (Sigma, St. Lois, MO).

Deposition of IgG was determined by using FITC-conjugated anti-mouse IgG antibody (Sigma,

St. Lois, MO). Negative controls were performed by substituting the primary antibody with an

irrelevant pre-immune serum.

Cell Culture: Human interstitial fibroblasts (TK173) were maintained in culture as

previously described (27, 32). For stimulation of fibroblasts, media were replaced with serum-

free Iscove�s modified Dulbecco�s medium (IMDM) (45).

Direct-enzyme-linked immunoabsorbent assay: We performed ELISAs for estimation

of type I collagen and fibronectin as described previously (43). Quiescent fibroblasts (8x103

cells/ well) were stimulated with rhBMP-7 (1.0, 10.0 and 100 ng/ml) in IMDM medium that was

supplemented with 50 mg/ml ascorbic acid and aminopropionitrile (Sigma, St. Louis) for 48 h.

Supernatants were analyzed in Maxisorp� (Nunc) plates using primary antibodies raised

against type I collagen (Southern Biotechnologies, Birmingham, AL), and fibronectin (Sigma, St.

Louis). All assays were performed in triplicate and repeated at least 3 times. The working range

was 10 to 1000ng/ml collagen type I and 0.1 to 10 mg/ml for fibronectin.

Zymography: Zymography was performed as described previously (57). Briefly, cells

(1x105 per well) were plated in 6-well plates and grown for 6 h in DMEM medium containing

10% FCS. The medium was replaced with IMDM or with IMDM containing 1, 10, or 100 ng/ml


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rhBMP-7. After incubation, medium was removed, cells were counted and the volume of

medium was normalized to cell counts. Electrophoresis was performed using 20 ml of medium

per lane in 10 % gelatin zymogram ready-cast gels (Bio Rad, Hercules, CA). Gels were washed

twice for 10 min at room temperature in renaturing buffer (2.5% v/v Triton X-100) and then

incubated for 18 h at 37 °C in development buffer (50 mm Tris, 200mm NaCl, 5 mm CaCl2, 0,02

% v/v Brij-35). Bands were visualized by staining the gel with Coomassie blue.

Statistical analysis. All values are expressed as mean + SEM unless specified.

Analysis of variance (ANOVA) was used to determine statistical differences between groups

using Sigma-Stat� software (Jandel Scientific, San Rafael, CA). Further analysis was carried

out using t test with Bonferroni correction to identify significant differences. A level of p<0.05

was considered statistically significant.

Results

Long-term treatment with rhBMP-7 does not induce significant side effects. rhBMP-7 is

currently used in the USA and Europe as an injectable drug to enhance bone formation when

applied locally to fractured areas. Hence, we performed initial four-month safety and toxicology

studies with rhBMP-7 administered intraperitoneally in normal mice (MRL/MpJ). After four

months of rhBMP-7 administration at three different doses, provided three times weekly, the

normal MRL/MpJ mice were sacrificed and various different tissues and body fluids were

examined for any toxic side effects. Histopathology from brain, heart, lung, liver, spleen, kidney

and muscle revealed insignificant toxicity. Ectopic bone formation at the injection site was not

observed. Only four out of 24 mice displayed protein casts in few medullary collecting ducts.


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One mouse from the 4-week/300mg/kg group also showed mild symptoms of hepatitis. Two

mice displayed mildly dilated lateral ventricles, a lesion that is usually detected as an incidental

finding in many mouse studies (Table 1).

Decrease of tubular endogenous BMP-7 in progression of renal disease in MRL/MpJlpr/lpr

and in Col4A3-/- mice. Recent studies suggested that renal injury was associated with a

decrease of endogenous BMP-7 expression and that administration of exogenous rhBMP-7

served to restore renal function and morphology (21, 52, 54, 56). In the present study we

attempted to explore the potential of rhBMP-7 to prevent progression of renal disease in genetic

models of renal disease. We first assessed endogenous BMP-7 in the normal and diseased

kidneys of these mice. Due to a mutation in the Fas gene MRL/MpJlpr/lpr mice develop a lupus

like disease with progressive renal fibrosis (23). While kidneys from MRL/MpJ control mice

displayed BMP-7 in tubular cells and in glomeruli, confirming previous observations, BMP-7 was

notably decreased in injured kidneys from MRL/MpJlpr/lpr mice (17, 54) (Figure 1 A,B).

Additionally, we used mice that lack the collagen type IV a3 gene (Col4A3-/-) as a second model

for progressive renal disease. Col4A3-/- mice develop progressive renal disease, which leads to

death between week 12 and 16 (1, 30). While C57BL/6 control mice displayed tubular and

glomerular BMP-7 localization, progression of renal disease in Col4A3-/- mice was associated

with a decrease in endogenous BMP-7 expression (Figure 1 C,D). We thus speculated that

supplementation of exogenous rhBMP-7 could improve renal pathology and function in both

these genetic mouse models for chronic renal disease.

rhBMP-7 ameliorates progression of chronic renal disease in MRL/MpJlpr/lpr mice.

To evaluate a potential role for rhBMP-7 in the treatment of chronic renal disease, we first


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utilized MRL/MpJlpr/lpr lupus nephritis mice (11). We initiated i.p. injection of rh BMP-7 at 4 weeks

of age. At this age, very early signs of disease could be demonstrated in these mice. After

termination of the study at 16 weeks of age, rhBMP-7 treated mice displayed reduced relative

interstitial volume as well as a reduced number of atrophic tubular structures when compared to

untreated control (Figure 2 A-D). Animals that were treated with 300 mg/kg rhBMP-7 also

displayed reduced glomerular crescents, markedly reduced glomerulosclerosis, and reduced

glomerular hypercellularity (Figure 2 E, F). Serum creatinine levels were significantly reduced in

mice that were treated with 300 mg/kg rhBMP-7 after four weeks of treatment, whereas by the

end of study results failed to reach significance due to wide variance in the treatment groups

(Figure 2G).

These findings also correlated with reduced interstitial staining for type I collagen in the

treated mice, compared to control mice (Figure 3 A, B). Localization for IgG in glomeruli did not

show substantial difference between untreated and treated mice (Figure 3 C, D).

Treatment with rhBMP-7 inhibits progression of renal disease in Col4A3-/- mice.

We next attempted to investigate, whether administration of exogenous rhBMP-7 had a similar

role on the progression of renal disease in Col4A3-/- mice. Treatment with rhBMP-7 was initiated

after six weeks of age until the study was terminated at the age of 14 weeks (30). In this study,

we treated mice with 300 mg/kg rhBMP-7, which was established by our previous study as the

optimum dosage, and was also confirmed by other groups (21, 52). Treatment with 300 mg/kg

rhBMP-7 led to reduction of tubulointerstitial fibrosis as determined by reduced relative cortical

interstitial volume and significantly reduced tubular atrophy (Figure 4 A-C). Reduction of renal

damage led to decreased renal pathology-related mortality rate in the rhBMP-7 treated group

(Figure 4C). Five out of seven untreated control mice died due to renal failure before week 14,


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whereas none of the treated mice died due to uremia and kidney disease. Treated mice lived up

to week 26. Renal function, as assessed by serum creatinine (Figure 4D), BUN (Figure E) and

urine protein (Figure 4F), revealed significant improvement in BMP-7 treated mice as compared

to untreated Col4A3-/- mice.

rhBMP-7 has anti-fibrogenic effects on renal fibroblasts. We next attempted to

evaluate the effect of rhBMP-7 on interstitial fibroblasts in order to gain further insights into the

anti-fibrotic action of rhBMP-7. Interstitial fibroblasts play a central role in the progression of

chronic renal disease as the main mediators of ECM deposition, the characteristic feature of

fibrosis (13, 39). We utilized a human interstitial fibroblast cell line (TK173), which displays an

activated fibroblast-like state in vitro, to evaluate the potential of rhBMP-7 to inhibit pro-fibrotic

contributions of these cells (27, 32). Synthesis of type I collagen and fibronectin are considered

as key features of activated fibroblasts and we show here that administration of rhBMP-7

decreases synthesis of collagen type I and fibronectin in the TK173 cells in a dose dependent

manner, without significant alteration in the proliferative capacity of these cells (Figure 5A,B)

(13, 39). In addition to increased synthesis, a decrease in ECM-degradation is considered

important for shifting the balance of ECM-homeostasis towards enhanced matrix deposition

during fibrogenesis (13, 44). Human interstitial fibroblasts secrete substantial levels of matrix

metalloproteinases, MMP-2 and MMP-9, which possess the capacity to cleave basement

membranes and interstitial ECM constituents (8, 10, 26, 48). Treatment of TK 173 cells with

rhBMP-7 led to up-regulation of MMP-2 and also of MMP-9 (Figure 5 C). In the Mrl/MpJlpr/lpr

mice, upregulation of MMP-2 by the interstitial fibroblasts by administration of rhBMP-7 was also

observed (Figure 5 D, E). While MMP-2 was absent in the interstitial areas containing abundant

myofibroblasts, MMP-2 was markedly increased in the interstitium of rhBMP-7 treated

Mrl/MpJlpr/lpr mice (Figure 5 D, E). In the rhBMP-7 treated kidneys, MMP-2 mainly co-localized


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with a-smooth muscle actin (a-SMA) expressing activated myofibroblasts, further suggesting a

potential of rhBMP-7 to induce MMP-2 in the interstitial fibroblasts (Figure 5 D, E). MMP-9 was

significantly present in the interstitium of untreated control kidneys and thus further increase of

MMP-9 could not be demonstrated following treatment with BMP-7 (Figure 5 F, G) .

Furthermore, MMP-9 in the interstitium rarely co-localized with a-SMA, suggesting that rhBMP-7

in interstitial fibroblasts activates mainly MMP-2 in the injured kidneys (Figure 5 F, G).

Discussion

Progression of chronic renal disease towards ESRF still represents one of the biggest

problems in nephrology, as it leads to an increasing number of patients who require long-term

renal replacement therapy, such as dialysis or kidney transplant (9, 35). While ACE-inhibitors

are currently the most promising therapeutic agents to inhibit progression of renal fibrosis in

clinical use, specific therapeutic options are still not available (2, 7, 15, 25, 55).

In this regard, several recent studies have demonstrated a beneficial role for

administration of exogenous rhBMP-7 in different animal models of chronic renal injury (21, 31,

52, 56). BMP-7 is a member of the TGF-b superfamily and has an important function during

kidney development (20). It is associated with condensation of the metanephric mesenchyme,

leading to the formation of tubules and glomeruli (20, 41). In the adult kidney, BMP-7 expression

can be detected in tubular epithelial cells and in podocytes (17, 54). Endogenous BMP-7

expression significantly decreases during acute renal injury and administration of exogenous

rhBMP-7 accelerates the repair of the injured kidney, suggesting that BMP-7 plays a role in the

maintenance kidney homeostasis (42, 49). Similarly, BMP-7 expression is decreased in several

induced animal models of chronic renal injury, and administration of exogenous rhBMP-7 in

these models inhibits progression or enables recovery of chronic renal injury (21, 52, 54, 56).


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In this regard, in the present study we establish for the first time a role of rhBMP-7 as a

therapeutic in two genetic mouse models, which mimic long-term chronic renal disease. In

MRL/MpJlpr/lpr mice, which develop lupus nephritis-like renal disease associated with significant

tubulointerstitial fibrosis after three months, treatment with rhBMP-7 inhibits progression of renal

disease in a dose dependent manner. In mice deficient in the a3 chain of type IV collagen,

ESRF associated with interstitial fibrosis after 14 weeks is observed. rhBMP-7 prevents renal

fibrosis and renal related mortality in these mice and increases their survival by 12 weeks. Thus,

we demonstrate the anti-fibrotic effect of rhBMP-7 in two long-term mouse models for chronic

renal fibrosis, providing further evidence for the use of rhBMP-7 as a therapeutic agent for

chronic renal injury.

Previous studies have suggested that rhBMP-7 exerts its anti-fibrotic effect mainly on

tubular epithelial cells, where it inhibits the release of pro-inflammatory chemokines (17). It is

also shown to reverse epithelial to mesenchymal transition (EMT), while acting as an antagonist

of transforming growth factor b1 (TGF-b1)-induced E-cadherin downregulation (56). In chronic

renal fibrosis, activated interstitial fibroblasts, which can either derive from resident interstitial

fibroblasts, from tubular epithelial cells via EMT or from bone marrow-derived mesenchymal

precursor cells, are considered the main pathogenic mediators of renal disease (13, 19, 22, 39).

Renal fibrosis is characterized by an excessive deposition of interstitial ECM, which results in

destruction of kidney architecture and impairment of renal function. Activated interstitial

fibroblasts are the main mediators of enhanced ECM synthesis (3, 6, 14, 16, 33). In the present

study we provide evidence that rhBMP-7 functions (in addition to its effects on tubular epithelial

cells) by inhibiting pro-fibrotic contributions of activated interstitial fibroblasts. We hypothesize

that up-regulation of MMP-2 by rhBMP-7 reflects increased ECM degradation and thus

potentially a decrease in scar tissue in the renal interstitium (46, 58). TGF-b1, the main pro-


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fibrotic growth factor involved in renal fibrogenesis, mediates all these three pro-fibrotic features,

which are inhibited by rhBMP-7 in our studies, in renal fibroblasts (6, 46). Previous studies have

also demonstrated the potential of rhBMP-7 to counteract TGF-b1 action in tubular epithelial

and mesangial cells (53, 56). Our research coupled with these previous important studies

suggest that rhBMP-7 functions on tubular epithelial cells, mesangial cells and fibroblasts to

restore the health of kidney tissue. These studies provide us further with confidence that

rhBMP-7 should be tested in human clinical trials involving kidney disease patients.


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Acknowledgements

This study was supported by a sponsored research project from Curis, Inc.; in part by

NIH grants DK 51711 and DK 55001, American Society of Nephrology Carl Gottschalk-

research award, grants from the Deutsche Forschungsgemeinschaft DFG Mü523/7-1 (to G.M.)

and DFG ZE5231/1 (to M.Z.).


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Abbreviations

BMP, Bone Morphogenic Protein; DMEM, Dulbecco�s Modified Eagle�s Medium; ECM,

extracellular matrix; EMT, epithelial-to-mesenchymal transition; ESRF. End Stage Renal Failure;

MMP, Matrix Metalloproteinase; PAS, Periodic Acid Schiff; STZ, streptozotocin; TGF,

Transforming Growth Factor; UUO, unilateral urethral obstruction.


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Figures

Figure 1. Immunofluorescence Staining. Frozen kidney sections were stained with

antibodies to BMP-7 and the staining was visualized using FITC-conjugated secondary

antibodies. (A) Normal kidney from Mrl/MpJ control mice displayed tubular staining (arrows) and

glomerular staining (arrowhead). (B) BMP-7 was decreased in kidneys from 16-week old

Mrl/MpJ lpr/lpr mice with interstitial fibrosis. (C) BMP-7 was present in kidneys from control

C57BL/6 mice. (D) In kidneys from 14-week old Col4A3-/- mice, severe interstitial fibrosis was

associated with decreased BMP-7 staining. Original magnification x200.

Figure 2. Treatment of MRL/MpJlpr/lpr mice with rhBMP-7 ameliorates progression of

chronic renal disease. (A, B) Light microscopy. The pictures display representative areas of

PAS-stained kidney sections from MRL/MpJlpr/lpr mice at the age of 16 weeks. Histology of

animals that were treated with 300 mg/kg rhBMP-7 revealed a drastic delay of disease

progression (A) compared to control animals (B). (C-F) Morphometric analysis. Kidney

sections were analyzed for the relative interstitial volume, tubular atrophy, glomerular cellularity

and crescent formation. The graphs summarize average results of each group. (C) Treatment

with rhBMP-7 resulted in a significantly decreased relative interstitial volume compared to

untreated mice. (48.4% +8.9). (D) Occurrence of atrophic tubules was significantly decreased in

the 300 mg/kg treated group (8.5% +7.3 of control). (E, F). Glomerular injury, as determined by

glomerular cellularity (68.5% +1.3) and formation of glomerular crescents (2.8% +0.35 of

control) was decreased in the 300 mg/kg treated group. (G) Serum creatinine levels. The graph

summarizes serum creatinine levels between week 4 and week 16. **=p<0.001, *=p<0.05.

Original magnification x200.


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Figure 3. Immunofluorescence Staining. Frozen kidney sections, which were obtained from

MRL/MpJlpr/lpr mice at the age of 16 weeks, were stained with antibodies for type I collagen and

IgG. The pictures display representative sections from untreated control mice and from mice

that were treated with 300 mg/kg rhBMP-7. (A, B) Interstitial staining for type I collagen (arrows)

was substantially decreased in rhBMP-7 treated MRL/MpJlpr/lpr mice compared to untreated

control (C, D). Staining for IgG deposition (arrows) did not reveal significant differences between

the two groups. Original magnification x200.

Figure 4. Treatment of Col4A3-/- mice with rhBMP-7 results in decrease of renal disease

and reduction of renal-related mortality. Col4A3-/- mice were treated with 300 mg/kg rhBMP-

7, control mice were injected with vehicle buffer alone. Urine and blood was obtained every two

weeks, the study was terminated at 14 weeks of age. (A, B) Light microscopy. Histology of

kidney specimens from Col4A3-/- mice revealed markedly improved appearing tissue in rhBMP-7

treated group, as compared to substantial tubulointerstitial fibrosis in untreated control Col4A3-/-

mice (C). Relative interstitial volume and tubular atrophy index were determined by

morphometric analysis. The graph summarizes average values of each group. Treatment with

300 mg/kg of rhBMP-7 resulted in decreased relative interstitial volume (23% +3.3 of control)

and tubular atrophy (7.7% +4.5 of control). Whereas 71% of untreated mice died before week

14 due to renal failure, no renal related deaths occurred in the BMP-7 treated group. (D, E, F)

Renal function parameters. Serum-creatinine, BUN and urine protein were determined every

other week. The graphs summarize results, which were obtained during the study. (***:

P<0.0001, *: P<0.05). Original magnification x200.


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Figure 5. BMP-7 has anti-fibrogenic properties on renal interstitial fibroblasts. (A)

Collagen ELISA. (A-C). TK 173 human interstitial fibroblasts were subjected to rhBMP-7 for 48

h. Tissue culture supernatants were evaluated for secretion of type I collagen and fibronectin by

ELISA and for release of MMPs by zymography. The graphs summarize three independent

experiments. (A) Collagen ELISA. rhBMP-7 significantly decreased rhBMP-7 secretion of type

I collagen into tissue culture supernatants (15.2% + 1.9 compared to control at 100 ng rhBMP-7/

ml). (B) Fibronectin ELISA. After 48 h, fibronectin was significantly decreased in tissue culture

supernatants of interstitial fibroblasts that were exposed to rhBMP-7 (27.1% + 5.1 compared to

control at 100 ng/ml). (C) Zymography. Interstitial fibroblasts were treated with BMP-7 for 48 h

and the tissue culture supernatants were analyzed by zymography for MMP-2 and MMP-9. The

picture displays a representative gel. Densitometric analysis revealed a significant increase of

MMP-2 (210.5% +10.27 compared to untreated control) and MMP-9 (151.2% +4.3) after

treatment with 100 ng/ml rhBMP-7 (D, E). MMP-2/ a-SMA double staining. Frozen kidney

section from rhBMP-7 treated and control MRL/MpJlpr/lpr mice were analyzed after termination of

the study at the age of 16 weeks. In immunohistochemistry double staining experiments for a-

SMA (FITC-green) and MMP-2 (Rhodamine-red), MMP-2 was absent in myofibroblasts after 16

weeks in untreated MRL/MpJlpr/lpr mice (arrows). Administration of exogenous rhBMP-7 resulted

in increased interstitial MMP-2 staining (arrowheads). (F, G). MMP-9/ a-SMA double staining.

Immunohistochemistry double staining experiments for a-SMA (FITC-green, arrows) and MMP-

9 (Rhodamine-red, arrowheads). Substantial staining for MMP-9 was present in untreated

MRL/MpJlpr/lpr mice, treatment with rhBMP-7 resulted in insignificant changes. Original

magnification x400. ***=p<0.0001, *=p<0.05.


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Table 1.

Group Protocol Histopathological Findings1 Vehicle buffer, start week 4 No histopathological abnormalities2 Vehicle buffer, start week 8 Protein casts in few collecting ducts in the

kidney in one mouse3 BMP-7, 30 mg/kg, start week 4 Protein casts in few collecting ducts in one

mouse4 BMP-7, 100 mg/kg, start week 4 One mouse with mild form of hepatitis5 BMP-7, 300 mg/kg, start week 4 Protein casts in few collecting ducts in two

mice, one mouse with mild form ofhydrocephalus

6 BMP-7, 30 mg/kg, start week 8 Protein casts in few collecting duct in onemouse

7 BMP-7, 100 mg/kg, start week 8 Protein casts in few collecting ducts in onemouse, mild hydrocephalus in one mouse

8 BMP-7, 300 mg/kg, start week 8 No histopathological abnormalities

Table 1. BMP-7 treatment of normal MRL/MpJ strain mice. Brain, lung, kidney, liver, spleen,

heart and skin were evaluated for histopathological abnormalities. A mild form of hydrocephalus

and incidental findings of mild hepatitis turn up in many mouse studies. This particular strain

was selected to serve as the most appropriate control for the MRL/MpJlpr/lpr lupus nephritis mice

used in the present study.


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Figure1


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Figure 2


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Figure 3


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Figure4


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Figure 5

Bone Morphogenic Protein7 Inhibits Progression of Chronic Renal Fibrosis Associated with two Genetic Mouse Models

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