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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Urol. Author manuscript; available in PMC 2012 August 13.
Published in final edited form as:
PMCID: PMC3418146
NIHMSID: NIHMS391343

The BMP-7–Smad1/5/8 Pathway Promotes the Repair of the Kidney Following Obstruction-Induced Renal Injury

Scott R Manson, Ph.D.,1 Robert A Niederhoff, Ph.D.,1 Keith A Hruska, M.D.,2 and Paul F Austin, M.D.1

Abstract

PURPOSE

Urinary tract obstruction causes hydroureteronephrosis and requires surgical intervention to prevent the development of permanent renal injury. While many studies have focused on the development of renal injuries, here we examine the molecular mechanisms that promote renal recovery following the correction of obstruction.

MATERIALS AND METHODS

A reversible murine model of ureteral obstruction was used to examine the BMP-7 and TGF-β signaling pathways during renal recovery following obstruction-induced injury. Analysis was conducted using standard molecular techniques including RT-PCR, ELISA, immunoblotting, and co-immunoprecipitation.

RESULTS

We found that the upregulation of BMP-7 following the correction of obstruction inhibits TGF-β-dependent pro-fibrotic pathways that are central to the pathogenesis of renal injury. The inhibitory effects of BMP-7 are mediated, in part, by the activation of its downstream target proteins, Smad1, Smad5, and Smad8, which suppress the activity of TGF-β-dependent Smad proteins and, in turn, inhibit the expression of TGF-β-dependent genes. Finally, the activation of the BMP-7–Smad1/5/8 pathway during renal recovery promotes the restoration of renal architecture and resolution of fibrosis in the kidney following the correction of obstruction.

CONCLUSIONS

Together, these findings demonstrate that the BMP-7–Smad1/5/8 pathway promotes the repair of the kidney following obstruction-induced injury. Accordingly, the BMP-7 pathway represents an important therapeutic target to stimulate the innate repair mechanisms of the kidney during the treatment of obstruction-induced renal injuries.

Keywords: Kidney, Ureteral Obstruction, Fibrosis, Bone Morphogenic Protein 7, Smad Proteins

INTRODUCTION

Obstructive uropathies are a leading cause of renal injury, chronic renal insufficiency, and renal failure.13 While current surgical approaches are frequently able to alleviate urinary tract obstructions, the possibility for irreversible renal injury remains even following treatment.2, 3 Therefore, the clinical management of obstructive uropathies would benefit from the development of therapeutic approaches that promote renal recovery following injury.

While the development of renal injuries is well understood,2, 3 renal recovery following injury is less clearly understood. Despite lacking a true regenerative ability,4 the mature kidney has an innate ability to restore renal structure and function following injury. However, this reparative ability is diminished following severe renal injury.5, 6 Understanding the processes that promote renal recovery and their molecular regulation may enable the development of therapeutic approaches to stimulate the innate repair mechanisms of the kidney during the treatment of obstruction-induced injuries.

At the molecular level, the activation of the TGF-β pathway plays a central role in the pathogenesis of renal injury by promoting apoptosis, epithelial-mesenchymal transformation, matrix protein synthesis, and other pro-fibrotic events that lead to the disruption of renal structure and function.7 Accordingly, neutralization of TGF-β inhibits the progression of obstruction-induced renal injury.810 Another member of the TGF-β superfamily, BMP-7, has the distinguishing property of inhibiting TGF-β-dependent biological functions.11 Both TGF-β and BMP-7 signal through receptor complexes that phosphorylate Smad transcription factors.1113 TGF-β and BMP-7 have different functions, in part, because they promote the activating phosphorylation of distinct Smad proteins. TGF-β activates Smad2/3 whereas BMP-7 activates Smad1/5/8. These phosphorylated Smad proteins then bind the Smad4 protein and regulate the transcription of target genes in a pathway-specific manner.1113 While the signaling pathways downstream of TGF-β and BMP-7 have been well defined, the mechanisms by which BMP-7 counters TGF-β in the kidney have not yet been clearly defined.

Importantly, treatment with exogenous BMP-7 inhibits the development of renal injuries.1416 In this manuscript, we explore the possibility that BMP-7 may inhibit the pathogenesis of renal injury, in part, by promoting the repair of the kidney. While several studies have demonstrated that exogenous BMP-7 reverses the progression of chronic renal injury, 17, 18 chronic injury models have not been able to effectively differentiate between the effects of BMP-7 on the development of renal injury and on the repair of renal injuries since, in these models, the injurious stimuli is continuously present.

In beginning to determine the role of the BMP-7 pathway in renal recovery following injury, several important questions remain to be answered: (1) Is the BMP-7 pathway regulated during renal recovery? (2) By what mechanisms does BMP-7 counter TGF-β in the injured kidney and are these counter-regulatory mechanisms subject to regulation during renal recovery? and (3) Is pharmacologic manipulation of the BMP-7 pathway an effective therapeutic approach to stimulate the innate repair mechanisms of the kidney during the treatment of renal injuries? Here, we begin to address these questions in order to better understand the molecular mechanisms that contribute to renal recovery following obstruction-induced injury.

MATERIALS AND METHODS

Reversible Unilateral Ureteral Obstruction

8–10 week old C57BL/6J mice were obstructed by placing a vascular clamp on the proximal ureter and, when indicated, the obstruction was reversed by subsequently removing the clamp.6 Mice were treated with either PBS or 300 μg/kg of purified BMP-7 (R&D Systems), as indicated.

Masson’s Trichrome Staining

Kidneys were fixed in Histochoice (Amresco). Slides containing 5 μm tissue slices were stained using Masson’s Trichrome Reagent (Richard Allen Scientific) according to product specifications.

Type IV Collagen Immunostaining

Slides were prepared (see above), subjected to antigen retrieval by boiling in 10 mM citrate buffer (pH 6.0), and stained using rabbit anti-Collagen IV/pro-Collagen IV (Millipore) and FITC-conjugated anti-rabbit antibodies (Sigma). Three photographs/sample were uniformly taken of the region bounded by the renal capsule and the cortico-medullary junction adjacent to the papilla at 200x magnification. Interstitial/tubular volume were visually quantified by overlaying a grid on slide photographs and counting the grid points located in the interstitial/tubular regions.19

Quantification of Total Kidney Collagen Content

Kidneys were hydrolyzed, resuspended in citrate-acetate buffer (pH 6.0), and oxidized with 0.35% Chloramine T. Hydroxyproline was quantitated by measuring the conversion of 7% dimethyl-aminobenzaldehyde to a colormetric product by hydrolyzates using spectrophotometry (558 nm) and comparison to hydroxyproline standards. Collagen content was determined using the approximation that collagen contains 14% hydroxyproline.20

RT-PCR

Kidneys were pulverized in liquid nitrogen, homogenized in Trizol (Invitrogen), and RNA was isolated according to product specifications. Semiquantitative reverse transcriptase PCR was conducted using Superscript RT-PCR (Invitrogen) for 24–32 cycles with the following primers: α-Smooth Muscle Actin: 5′-ctgagcgtggctattccttc-3′; 5′-gggggccaccctataataaa-3′ Collagen α1(I): 5′-actggtacatcagcccgaac-3′; 5′-ggtggagggagtttacacga-3′ GAPDH: 5′-actccactcacggcaaattc-3′; 5′-ccttccacaatgccaaagtt-3′

ELISA

Kidneys were pulverized in liquid nitrogen and homogenized in 20 mM Tris-HCl, pH 7.5, 2M NaCl, 0.1% Tween, and 1 mM EDTA. Protein levels were determined using a BMP-7 ELISA kit (R&D Systems) according to product specifications.

Immunoblotting

Kidneys were pulverized in liquid nitrogen and lysed in 83.3 mM Tris, 150 mM NaCl, 4% SDS, and 100 mM DTT supplemented with Protease/PhosSTOP inhibitors (Roche). Immunoblotting was conducted using rabbit anti-phosho-SMAD 2/3 (Cell Signaling Technologies), rabbit anti-phosho-SMAD 1/5/8 (Santa Cruz), rabbit anti-SMAD4 (Santa Cruz), or mouse anti-GAPDH (Chemicon), and either a HRP-goat anti-rabbit or HRP-goat anti-mouse secondary antibody (Jackson Immunoresearch).

Co-Immunoprecipitation

Kidneys were pulverized in liquid nitrogen and lysed in 50mM Tris-HCl, 150mM NaCl, 1 mM EDTA, 1% Triton, 1% sodium deoxycholate, and 0.1% SDS supplemented with Complete Protease/PhosSTOP phosphatase inhibitors (Roche). Lysates were immunoprecipitated with rabbit anti-SMAD4 (Santa Cruz) conjugated to Protein G beads (Dynal). Immunoblotting was conducted (as above).

Statistical Analysis

Data are presented as mean values ± standard deviation. Statistical significance was analyzed by ANOVA with Bonferroni correction.

RESULTS

Reversible Unilateral Ureteral Obstruction (UUO) as a Model of Renal Recovery From Obstruction-Induced Injury

To better understand the mechanisms that contribute to renal recovery from obstructive uropathies, we studied a model of acute renal injury in response to unilateral ureteral obstruction (UUO).6 Importantly, in this model, the subsequent reversal of the obstruction mimics the surgical correction of obstructive uropathies in patients and serves as a model of the recovery of the kidney from acute obstruction-induced injury.

Obstructive uropathy potentially results in decreased renal function through the disruption of renal architecture and the promotion of renal fibrosis.2, 3 Indeed, these deleterious changes our effectively reproduced in our model, as UUO causes renal fibrosis (Figure 1A) characterized by interstitial expansion (Figure 1B, P<0.01), loss of tubular volume (Figure 1C, P<0.01), and accumulation of collagen (Figure 1D, P<0.005).

Figure 1Figure 1Figure 1
The Recovery of the Kidney Following Obstruction-induced Renal Injury

In using the UUO model to examine renal recovery, we found that mice that undergo two days of UUO develop renal fibrosis but, following reversal of the obstruction and a recovery period, much of the renal damage subsides (Figure 1A). Reversal of the obstruction promotes a decrease in interstitial volume (Figure 1B, P<0.005), an increase in tubular volume (Figure 1C, P<0.01) and a decrease in collagen (Figure 1D, P<0.005). These findings demonstrate that the resolution of fibrosis and the restoration of renal architecture are processes that contribute to renal recovery. We next sought to examine the molecular regulation of these innate repair mechanisms.

The Activation of the BMP-7–Smad1/5/8 Pathway During Renal Recovery Inhibits TGF-β-Dependent Pro-Fibrotic Pathways in the Injured Kidney

We hypothesized that BMP-7 plays a role in renal recovery following obstruction-induced injury. In examining this possibility, we found that, while BMP-7 levels are downregulated following UUO, BMP-7 levels are restored following the reversal of UUO (Figure 2A, P<0.005). Similarly, we found that the upregulation of BMP-7 is associated with the restoration of the activity of its target proteins, Smad1/5/8 (Figure 2B). Together, these findings demonstrate that the BMP-7–Smad1/5/8 pathway is activated during the repair of obstruction-induced injuries.

Figure 2
The BMP-7–Smad1/5/8 Pathway is Activated During Renal Recovery Following Obstruction-Induced Renal Injury

Although the BMP-7 pathway is activated during renal recovery, the possibility remained that the activation of the BMP-7 pathway is merely a consequence of renal recovery itself. To determine the functional importance of BMP-7, we examined the molecular consequences of the activation of the BMP-7 pathway in the injured kidney. We found that, treatment with exogenous BMP-7 inhibits the transcription of several TGF-β-dependent genes that are central to the pathogenesis of renal injury and that are normally increased in the injured kidney including collagen α1(I) (a gene that encodes a protein that is a significant contributor to fibrosis21) (P<0.05) and α-smooth muscle actin (a gene that encodes a protein that contributes to the differentiation of pro-fibrotic myofibroblasts22) (P<0.005) (Figure 3).

Figure 3
BMP-7 Suppresses TGF-β-dependent Pro-Fibrotic Pathways in the Injured Kidney

We next sought to determine the mechanisms by which BMP-7 inhibits the activity of TGF-β-dependent pathways. We found that exogenous BMP-7 promotes the formation of BMP-7-regulated Smad1/5/8–Smad4 transcription factor complexes and reduces the obstruction-induced formation of TGF-β-regulated Smad2/3–Smad4 transcription factor complexes in the injured kidney (Figure 4A) despite the continued presence of elevated levels of activated/phosphorylated Smad2/3 proteins (Figure 4B) and TGF-β (data not shown). Together, these findings demonstrate that the activation of the BMP-7–Smad 1/5/8 pathway during renal recovery inhibits the activity of TGF-β-dependent Smad proteins in the injured kidney.

Figure 4
The BMP-7–Smad1/5/8 Pathway Suppresses the Formation of TGF-β-dependent Smad4 Transcription Factor Complexes

BMP-7 Promotes the Resolution of Fibrosis and the Restoration of Renal Architecture During the Repair of Obstruction-Induced Renal Injuries

Given that the upregulation of BMP-7 during renal recovery suppresses TGF-β-dependent pathways that are central to the pathogenesis of renal injury, we hypothesized that these molecular mechanisms contribute to the repair of renal injuries. Accordingly, we examined the effects of augmenting BMP-7 activity following the correction of obstruction. We found that, while mice that undergo two days of UUO followed by correction of the obstruction and a three day recovery period exhibit a 31.2% reduction in renal collagen during the recovery period, mice that are treated with exogenous BMP-7 during the recovery period exhibit a 53.1% reduction in renal collagen (Figure 5A, P<0.05). Similarly, BMP-7 treatment during recovery promotes the restoration of renal architecture by stimulating a 59.7% reduction in interstitial volume (compared to a 34.9% reduction in untreated mice) (Figure 5B, P<0.01) and a 63.0% reduction in the loss of tubular volume (compared to a 34.5% reduction in untreated mice) (Figure 5C, P<0.01). Together, these findings demonstrate that the activation of the BMP-7 pathway during renal recovery stimulates the restoration of renal architecture and the resolution of fibrosis in the kidney. Furthermore, it is notable that the renal protective effects of treatment with BMP-7 during recovery are only minimally improved by treatment with BMP-7 during both obstruction and recovery (Figures 5A–5D). Accordingly, these findings demonstrate that the renal protective effects of BMP-7 during the repair of established renal injuries may exceed even those of BMP-7 during the development of renal injuries.

Figure 5
BMP-7 Promotes the Repair of the Kidney Following Obstruction-Induced Renal Injury

DISCUSSION

An important long-term goal in the treatment of obstructive uropathies is the development of therapeutic approaches to stimulate the regression of fibrosis, repair of structural damage and, ultimately, recovery of renal function following obstruction-induced injury.13 However, little progress has been made towards achieving this goal, in part, because the processes that promote renal recovery following injury are poorly understood.5

Over the course of this study, we found that the BMP-7–Smad1/5/8 pathway is activated during the repair of the kidney following acute, reversible obstruction-induced injury. Furthermore, the activation of the BMP-7 pathway promotes the resolution of fibrosis and the restoration of renal architecture during renal recovery. While BMP-7 has previously been reported to prevent the development of renal injury,1416 our studies demonstrate that BMP-7 has renal protective effects that extend beyond preventing the development of renal injury per se since BMP-7 is also able to stimulate the repair of established renal injuries following the correction of obstruction. Interestingly, in our model, the renal protective effects of treatment with exogenous BMP-7 during recovery are only minimally improved by treatment with exogenous BMP-7 during both obstruction and recovery. Given the absence of a synergistic effect, these findings suggest that the repair-promoting functions of BMP-7 may have played a significant role in previous studies examining the renal protective effects of BMP-7 during the development of obstruction-induced renal injury.1416

At the molecular level, the restoration of BMP-7 levels following the correction of obstruction is associated with the activation of its downstream target proteins, the Smad1/5/8 proteins. The activation of BMP-7-dependent Smad proteins opposes TGF-β-dependent pathways in the injured kidney by inhibiting the formation of TGF-β-dependent Smad2/3–Smad4 transcription factor complexes and the transcription of TGF-β-dependent genes (summarized in Figure 6). Given that TGF-β-dependent pathways are required for the pathogenesis of renal injury, 710 these findings suggest that the activation of BMP-7-dependent Smad proteins plays an important role in renal recovery following injury and in the renal protective effects of BMP-7.

Figure 6
A Model of BMP-7-Mediated Suppression of TGF-β-Dependent Pro-Fibrotic Pathways During the Repair of Renal Injuries

Nonetheless, the precise molecular mechanisms downstream of BMP-7 that promote renal recovery remain to be elucidated in future studies. It is likely that at least some of the repair-promoting functions of BMP-7 may be attributed to its ability to counter TGF-β. For example, BMP-7 may oppose TGF-β-induced matrix protein synthesis, protease inhibition, and myofibroblast recruitment and differentiation.7, 23 Other repair-promoting functions of BMP-7 may be attributed to functions independent of TGF-β, such as its ability to act as a renal morphogen.14 It has been shown that BMP-7 is indispensable for kidney development during embryogenesis14, 23 and, in turn, that tissue repair and the replacement of damaged cells through regeneration involve processes analogous to embryogenesis.24 Along with identifying the downstream molecular mechanisms that contribute to BMP-7-stimulated kidney repair, important future goals will be to determine why these pathways are impaired in prolonged obstructions that lead to permanent renal injury and also to develop therapeutic approaches to reactivate these innate repair mechanisms in the kidney during the treatment of obstructive uropathies.

CONCLUSIONS

As we work towards a better understanding of renal recovery following injury, reversible models of acute renal injury will serve as invaluable tools in elucidating the innate repair mechanisms of the kidney. Here, we demonstrate that the activation of the BMP-7–Smad1/5/8 pathway promotes the resolution of fibrosis and the restoration of renal architecture during renal recovery following the reversal of obstruction. Together, these findings demonstrate that the BMP-7–Smad1/5/8 pathway plays an important role in the repair of renal injuries that result from obstructive uropathies. Accordingly, the BMP-7 pathway represents a potential target of adjuvant therapy to stimulate the innate repair mechanisms of the kidney during the treatment of obstructive uropathies.

Acknowledgments

Sources of Financial Support:

National Institutes of Health and Midwest Stone Institute

Key of Definitions for Abbreviations

BMP-7
Bone Morphogenic Protein 7
REC
Recovery
SMAD
SMA and MAD-related protein
TGF-β
Transforming Growth Factor β
UUO
Unilateral Ureteral Obstruction

Footnotes

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