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Division of Nephrology, Harbor-UCLA Research and Education Institute, Inc., and UCLA, Torrance, California.
Correspondence to Dr. Raimund Hirschberg, Division of Nephrology, C-1-A, Harbor-UCLA Research & Education Institute, 1124 West Carson Street, Torrance, CA 90501. Phone: 310-222-3891; Fax: 310-782-1837; E-mail: rhirschberg{at}rei.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 III mRNA levels. In conclusion, in experimental diabetic
nephropathy, renal tubular BMP7 and some of its receptors decreased and
gremlin, a secreted BMP antagonist, increased. Some, but not all, of these
changes are explained by increased TGF-ß. The loss of BMP7 activity
per se is profibrogenic in tubular cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BMP7 (formerly called osteogenic protein-1 or OP1) is a homodimeric member of the BMP subfamily within the TGF-ß superfamily of cysteine-knot cytokines (6,7). In the adult organism, BMP7 is primarily expressed in kidney tubules, and lesser amounts are found in ovary, nonpregnant endometrium, and growth plates of cartilage (8,9).
BMP7 receptors are type I/II heterodimeric serine/threonine kinase receptors (10). Several different type I receptors, namely the activin receptor—like kinases (Alk) Alk2 (activin receptor I), Alk3 (BMP receptor IA), and Alk6 (BMP receptor IB) associate with the BMP type II receptor (BMPRII). Receptor activation induces intracellular recruitment and serine/threonine phosphorylation of smad substrates. Smad1, -5, and -8 are BMP-restricted smads, and smad1 and/or -5 are signaling substrates for BMP7 in different cell types (10,11,12). TGF-ß—restricted smad2 and -3 are not activated by BMP7.
The bioactivity of BMP7, like that of other TGF-ß superfamily members, is further regulated by a series of secreted, extracellular proteins that bind and neutralize peptide activity. Secreted BMP7 antagonists include noggin, follistatin, and gremlin (10,13,14,15,16,17).
We tested the hypothesis that loss of BMP7 activity is profibrogenic in proximal tubular cells. Moreover, studies were performed to examine whether, during the evolution of experimental diabetic nephropathy, renal tubular levels of BMP7 decrease and the expression of BMP7 receptors and secreted antagonists changes in favor of reduced BMP7 bioactivity. In vitro studies were performed to examine potential mechanisms of these changes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sprague-Dawley rats were made diabetic with a single intravenous injection
of streptozotocin, 65 mg/kg (Sigma, St. Louis, MO). At 2 d, glucose was
measured in tail blood with a glucometer, and animals with serum glucose
levels 
300 mg/dl were included. Rats were given NPH insulin
subcutaneously, to maintain serum glucose at 300 to 350 mg/dl. Control animals
did not receive streptozotocin or insulin.
In the first series, animals were killed at 30 wk. Kidneys were removed and
1-mm coronal sections were fixed in 4% paraformaldehyde and embedded in
paraffin. In the second series of rats, at 15 wk, urine was collected on ice
for 12 h to determine BMP7 excretion. Kidneys were removed, and 1-mm
coronal sections were fixed and embedded; total RNA was extracted with
RNA-Stat-60 (Tel Test, Friendswood, TX) from aliquot kidney sections.
Immunohistology for BMP7
Deparaffinized 5-µm sections were quenched, blocked, and incubated with
anti-BMP7 antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) and
biotin-labeled second antibody and visualized with streptavidin-horseradish
peroxidase (HRPO) and a substrate reaction. For control, the primary antibody
was replaced with nonimmune serum. Sections were lightly counterstained with
hematoxylin. Slides were examined with light microscopy, and BMP7-positive
tubules were counted by use of a point-grid. Quantitative data were derived by
dividing the point counts on BMP7-positive tubule cross-sections by the number
of points on any tubule cross-section multiplied by 100, to express results as
a percentage (19). In
addition, immunohistology for TGF-ß was also performed.
Urinary BMP7
Urine aliquots corresponding to 5% of excretion from three control and four
diabetic rats, respectively, were diluted with sterile water and concentrated
in spin concentrators (Millipore, Bedford, MA). Retentates were taken up in
nonreducing sodium dodecyl sulfate—polyacrylamide gel electrophoresis
sample buffer, electrophoresed in 15% gels, and transferred onto
nitrocellulose. Membranes were blocked and blotted successively with anti-BMP7
(1:1000), biotinylated second antibody, and HRPO-streptavidin. Bands were
visualized with chemiluminescence (Amersham, Arlington Heights, IL).
mRNA Levels Encoding BMP7, Alk2, Alk3, Alk6, BMPRII, Gremlin,
Follistatin, and Noggin
mRNA levels encoding BMP7, BMP receptors, and BMP antagonists were measured
by quantitative reverse transcription—PCR (RT-PCR). Random-primed
first-strand cDNA was synthesized from RNA with a commercial procedure
(Boehringer Mannheim, Indianapolis, IN). Aliquots of cDNA were amplified with
specific primers for each of the mRNA species
(Table 1), and 18S cDNA was
coamplified as the internal standard in each tube with specific primers and a
compatimer (Ambion, Inc., Austin, TX). Temperature cycling, cycle number, and
compatimer concentrations were optimized for each mRNA species. PCR products
were electrophoresed in 4% agarose gels that contained 0.5 µg/ml of
ethidium bromide. Resolved gels were photographed with Polaroid negative film
(Polaroid, Cambridge, MA). Bands were scanned by densitometry, and
densitometric units were expressed relative to the 18S mRNA band in the same
sample as a percentage of control.
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In vitro Studies
Experiments were performed by use of murine proximal tubular cells (mPTC)
that were derived from S3 segments of mouse proximal tubules by
microdissection
(18,20).
Cells were grown in Petri dishes or 6-well plates in Dulbecco's modified
Eagle's medium/F12 that contained insulin, transferrin, selenite, and 10%
fetal calf serum. At 95% confluence, cells were growth arrested in
serum-free medium that contained 0.1% bovine serum albumin for 24 h before
individual experiments.
Effects of Neutralization of BMP7 on Matrix Protein Expression in
mPTC
Because increased expression of interstitial extracellular matrix proteins
has previously been shown to be an abnormal function of tubular cells in
diabetic nephropathy, experiments were performed to examine whether
neutralization of BMP7 activity in mPTC is associated with changes in
fibronectin and/or collagen 1III expression. BMP7 was
neutralized with a recombinant fusion protein that consisted of the
high-affinity extracellular domain of Alk6 fused to the Fc region of IgG.
Confluent, arrested cells in 6-well plates were incubated without (control) or
with the Alk6/Fc chimera protein (R&D Systems, Minneapolis, MN), 2
µg/ml, for 72 h, and media were changed at 24 and 48 h to maintain high
levels of active Alk6/Fc-chimeras, (n = 6 each). Cells were then
washed, and total RNA was extracted from each well. mRNA encoding fibronectin
and collagen 1III were measured by quantitative RT-PCR.
BMP7 Cell Signaling in mPTC
Studies were performed to examine the preferred, BMP7-activated smad
substrate in mPTC and to examine whether BMP7 activates extracellular
signal-regulated kinase-1 and/or -2 (Erk1/2).
Smad Phosphorylation
Confluent, growth-arrested proximal tubular cells in a 6-well plate were
incubated with 1 nM rhBMP7 (kind gift from Kuber Sampath, Creative
Biomolecules, Boston, MA) (dissolved in 24 mM Na acetate [pH 4.5] containing
1% mannitol), or with 1 nM rhTGF-ß or both for 6 h. Media were removed,
and cells were lysed with lysate buffer (phosphate-buffered saline [pH 7.4]
that contained 1% Nonidet P-40, 0.5% Na-deoxycholate, 0.1% sodium dodecyl
sulfate, 100 µg/ml phenylmethylsulfonyl fluoride, 30 µl/ml aprotinin
[Sigma No. A6279], 100 µM of Na-orthovanadate, 2 µM of leupeptin, and 4
µM of pepstatin A), 1 ml/well. Lysates were cleared by centrifugation.
Immunoprecipitations were performed with an antibody that recognizes smad1 and
-5 (Santa Cruz Biotechnology) and protein Gplus-agarose overnight.
Immunoprecipitates were washed four times with lysate buffer, and pellets were
electrophoresed in a 10% minigel. Separated proteins were transferred onto
nitrocellulose and immunoblotted with anti-phospho-serine antibody (1:300,
Zymed, South San Francisco, CA), biotinylated second antibody, and
HRPO-streptavidin. Bands were visualized by chemiluminescence and captured on
x-ray film.
In separate experiments, confluent, growth-arrested mPTC in 10-cm culture plates were phosphate-depleted with P-free medium for 30 min and then metabolically labeled with 200 µCi/ml 32P-orthophosphoric acid (ICN, Irvine, CA) for 75 min. Subsequently, 1 nM rhBMP7 or BMP7-free diluent (control) were added for a further 45 min. Cells were washed twice with ice-cold medium and lysed. Lysates were immunoprecipitated with anti-smad1/5 antibody and electrophoresed, and the dried gel was autoradiographed. In a parallel experiment, in cells that were not P-labeled, immunoprecipitates underwent Western blot analysis with either specific anti-smad1 or anti-smad5 antibody (Santa Cruz Biotechnology).
Erk Phosphorylation
TGF-ß has been reported to activate Erk1 and -2 in certain cells,
including epithelial cells
(21). Hence, studies were
performed to examine whether BMP7 alters TGF-ß—induced Erk
phosphorylation. In these experiments, mPTC in 6-well plates were incubated
with rhTGF-ß1 (R&D Systems) or rhBMP7, or both, each at 1
nM, for 30 min. Cells were washed two times with ice-cold medium. Equal
aliquots of cell lysates were electrophoresed in 12% sodium dodecyl
sulfate—polyacrylamide gel electrophoresis gels and transferred onto
nitrocellulose. Membranes underwent Western blot analysis either with antibody
specific for phosphorylated Erk or with anti-Erk antibody (Santa Cruz
Biotechnology) and HRPO-labeled second antibody, and bands were visualized by
chemiluminescence.
Effects of TGF-ß1 on BMP7, Alk2 and -3, Gremlin, and
Follistatin Expression in mPTC
Cells that had been grown to confluence in 6-well plates and that were
growth arrested were incubated with or without 1 nM rhTGF-ß1
for 24 h (n = 6 each). Total RNA was extracted, and mRNA encoding
BMP7, Alk2, Alk3, gremlin, or follistatin were measured by quantitative
RT-PCR, as described above.
Effects of BMP7 on TGF-ß Expression in mPTC
To examine the question of whether BMP7 reduces TGF-ß expression in
cultured proximal tubular cells, mPTC in 6-well plates were incubated with the
peptide at 500 pM and 5 nM for 24 h (n = 6 each). Expression of
TGF-ß was measured by quantitative RT-PCR.
Statistical Analyses
Data are expressed as mean ± SEM. Statistical comparisons were made
by ANOVA, followed by the Newman-Keuls Multicomparison test with the use of
Stat-Most 2.5 software (Data Most Corporation, Salt Lake City, UT). P
< 0.05 was defined as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The decrease in tubular BMP7 observed immunohistologically in diabetic rats at 15 wk is associated with decreased urinary excretion of the protein (Figure 2). Moreover, renal BMP7 mRNA levels are also decreased at 15 wk of diabetic rats compared with those of controls (Figure 3).
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In contrast to controls, where immunoreactive TGF-ß is only found in glomerular arterioles but not in any other glomerular or in any tubular location, tubular expression of TGF-ß is seen in proximal as well as distal tubular segments at 15 wk, and more so at 30 wk, of diabetes (data not shown), as has also been described by other investigators (22,23,24,25).
Renal BMP7 Receptors Tend to Decrease in Diabetic Nephropathy. mRNA levels encoding Alk2, Alk3, and Alk6, the BMP-type I receptors that bind BMP7 with high affinity, and BMPRII mRNA levels were examined in control and diabetic rat kidney. Renal Alk2 and BMPRII mRNA levels were significantly decreased at 15 wk of diabetes, compared with those in timed controls (P < 0.05), and Alk3 and Alk6 mRNA tended to be reduced, albeit not significantly (Table 2).
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Secreted BMP Antagonists. Noggin mRNA is not found in normal or diabetic rat kidneys but is abundantly expressed in rat liver. Follistatin is expressed in both control and diabetic rat kidney, and mRNA levels at 15 wk were similar in both groups (Table 2). In contrast, compared with controls, renal gremlin mRNA levels were significantly increased at 15 wk of diabetes (Table 2).
In Vitro Findings
Neutralization of Endogenous BMP7. Incubation of mPTC with an
Alk6/Fc chimera protein that binds BMP7 with high affinity and neutralizes its
bioactivity significantly increased the expression of fibronectin by 35%
(P < 0.05, Figure
4). Collagen 1III mRNA levels tended to
increase, albeit not significantly (Figure
4).
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BMP7 Signaling in Proximal Tubular Cells. Immunoprecipitation of
lysates from mPTC with an antibody that recognizes smad1 and -5 demonstrates a
single band upon Western blot analysis of precipitates with
anti-phospho-serine antibody. This band is enhanced in cells exposed to BMP7,
compared with controls (lane 3 in Figure
5A). Serine phosphorylation of this 50 kD smad protein is not
increased in cells exposed to equimolar TGF-ß (lane 2 in
Figure 5A). Moreover,
TGF-ß does not alter the BMP7-induced phosphorylation (lane 4 in
Figure 5A). Immunoprecipitation
of 32P-labeled cell lysates and Western blot analysis confirms that
BMP7 phosphorylates either smad1 or -5 in proximal tubular cells
(Figure 5B). In Western blot
analysis, the phosphorylated band reacts with anti-smad5
(Figure 5C) but not anti-smad1
(blot not shown). These studies indicate that the exclusive or preferred smad
signaling substrate for BMP7 in proximal tubular cells is smad5. Moreover,
phosphorylation of smad5 by BMP7 is not inhibited by coincubation with
TGF-ß.
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BMP7 does not antagonize TGF-ß—induced phosphorylation of Erk1/2. In fact, in mPTC, BMP7 strongly activates Erk1 and -2 (Figure 6). Coincubation of cells with BMP7 and TGF-ß causes additive Erk phosphorylation (Figure 6). Neither TGF-ß nor BMP7 increase Erk1/2 protein levels (Figure 6).
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TGF-ß Down-Regulates BMP7 and Modifies Expression of BMP7 Receptors and Antagonists. In cultured proximal tubular cells, rhTGF-ß1 reduces the expression of BMP7 to 49 ± 3% of control levels (P < 0.05). TGF-ß also significantly down-regulates expression of Alk3 in proximal tubular cells to 51 ± 8% of control levels (P < 0.05) but does not affect Alk2 mRNA levels. Alk6 mRNA could not be detected im mPTC. In proximal tubular cells, follistatin mRNA levels were similar in controls and in cells that were incubated with TGF-ß. Gremlin mRNA is up-regulated by TGF-ß 2.4 ± 0.2—fold compared with control levels (P < 0.05).
BMP7 Does Not Affect TGFß Expression. Although TGF-ß down-regulates BMP7 mRNA levels in tubular cells, the reverse experiment is negative. rhBMP7 at either 0.5 or 5 nM does not affect expression of TGF-ß.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Renal tubular BMP7 expression decreases early in the course of experimental diabetic nephropathy; subsequently, BMP7 disappears almost completely from the kidney. Further-more, other regulators of BMP7 bioactivity are also changed during the evolution of experimental diabetic nephropathy. These include decreased renal expression of BMPRII and of some of the type I receptors and increased expression of secreted BMP antagonists. Overall, these findings provide evidence that, in addition to reduced BMP7 expression and protein levels, its biologic activity in the kidney is further reduced at the receptor and antagonist level.
These changes in renal BMP7 levels and the expression of its receptors and antagonists tend to coincide with the expression of TGF-ß, giving rise to the possibility that TGF-ß down-regulates BMP7, and this may be a major mechanism of the action of TGF-ß. Alternatively, loss of BMP7 could trigger the increase in TGF-ß expression during the evolution of diabetic nephropathy.
In cultured tubular cells, as shown in this study, TGF-ß decreases BMP7 expression, which suggests that a rise in tubular TGF-ß levels during the evolution of diabetic nephropathy contributes causally to the loss of BMP7. The contrary, BMP7-induced suppression of TGF-ß, which would suggest that the loss of BMP7 may cause a rise in TGF-ß, is unlikely, given that the respective experiment in cultured tubular cells is negative. TGF-ß also reduces or tends to reduce the expression of BMP7 type I and II receptors in cultured tubular cells. This may suggest that TGF-ß causes respective changes in renal receptor expression during the evolution of diabetic nephropathy in vivo.
Only one of the three BMP antagonists, gremlin, increases in diabetic rat kidneys, as has recently been shown by other investigators (31), as well as in this study. The present in vitro findings suggest that TGF-ß causes or contributes to the increase in gremlin. Increased gremlin will likely contribute to a further decrease in BMP7 bioactivity beyond that caused by the decreased expression of the protein itself, because it neutralizes BMP7 activity by binding the protein in the extracellular environment (13,15).
In proximal tubular cells, BMP7 activates smad5 but apparently not (or only minimally) smad1. The current experiments have also shown that TGF-ß does not antagonize the BMP7-induced serine phosphorylation of smad5. The specificity of BMP signals appears to be determined, in part, by the type I receptors (10). High-affinity BMP7 type I receptors that are expressed in tubular cells include Alk2 and -3, and activation of smad5 can be mediated through both of these type I receptors as well as through Alk6 (10,11,12).
BMP7 repidly phosphorylates Erk1 and -2 in proximal tubular cells, as shown in these experiments (Figure 6). TGF-ß also activates Erk1 and -2 in proximal tubular cells, as shown in this study (Figure 6) as well as by different investigators in other cell types (21). Activated Erk inhibits nuclear translocation of smad2 and -3 by phosphorylation in the linker region and, hence, reduces these TGF-ß signals (32). One mechanism by which BMP7 could inhibit TGFß-induced increased production of extracellular matrix (33) could be the activation of Erk1/2 by BMP7.
In summary, endogenous BMP7 prevents increased expression of interstitial extracellular matrix proteins in proximal tubular cells, and neutralization of BMP7 is in itself a profibrogenic event. During the evolution of experimental diabetic nephropathy, tubular BMP7 expression and expression of BMP7 receptors decreases, and secreted BMP antagonists increase. Some of these changes are induced by TGF-ß.
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Acknowledgments |
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References |
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) is enhanced by BMP7 but not by TGF-ß,
and TGF-ß does not reduce BMP7-induced serine phosphorylation of this
smad. (B) Cells were incubated with or without BMP7 after metabolic labeling
with 32P, immunoprecipitated with anti-smad1/5 antibody, and electrophoresed.
Autoradiography visualizes a single BMP7-phosphorylated band. (C) Western blot
analysis of immunoprecipitates similar to that in panel B, with antibody that
recognizes smad5 but not smad1 and identifies the phosphorylated band in panel
A and as smad5. Western blot analysis with anti-smad1 antibody was not
reactive (not shown).