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Los Angeles Biomedical Research Institute (LABioMed) at Harbor-UCLA Medical Center, Torrance, California
Submitted 15 April 2007 ; accepted in final form 4 September 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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(TGF-)]. HG as well as TGF- raise caspase-3 activity, increase apoptosis, and reduce cell survival which is, in part, blocked by BMP7. Knockdown and forced expression studies indicate that smad5 is required as well as sufficient for these actions of BMP7. These findings indicate that BMP7 is a differentiation and survival factor for podocytes, requires smad5, and can reduce diabetic podocyte injury. diabetic nephropathy; apoptosis; smad; caspase-3
(TGF-) with downstream involvement of p38 and smad7 (19, 25).
Bone morphogenetic protein-7 (BMP7) is a member of the BMP family within the TGF- superfamily of cysteine-knot growth factor/cytokines. BMP7 is required during kidney and ocular development and is developmentally expressed in many tissues including podocyte precursor cells (8). In adult mammalian organisms, BMP7 expression decreases or disappears from most tissues with some exceptions, notably the kidneys where BMP7 remains expressed in distal tubules and collecting ducts within the inner cortex and the cortico-medullary junction. In vivo in adult rodent glomeruli, BMP7 is exclusively expressed in podocytes but BMP7-binding receptors are expressed in podocytes as well as by other glomerular residential cells (3, 9, 13, 22, 28). In pluripotent cells BMP7 contributes to lineage differentiation and appears to participate in the induction and maintenance of a differentiated cell phenotype (8, 15).
As a protein family, BMPs utilize heterodimeric cell surface receptors for signal induction including the type I receptors activin-like kinase (Alk)-2, -3, and -6 and the type II receptors BMPRII and ActRIIA. Activation of BMP receptors phosphorylates the receptor substrates smad1 and smad5 (and/or smad8), which heterodimerize with the common smad4 to undergo nuclear translocation and transcriptionally regulate specific gene targets together with other coregulating proteins (7, 21). Similar to TGF-, BMPs can also regulate cell type and context, dependently other signals including the tyrosine kinases Erk, JNK, and p38.
Previous studies partly from this laboratory showed that BMP7 opposes profibrogenic events that occur downstream of TGF- in some cell types such as mesangial and proximal tubular cells (29, 34). Moreover, transgenically expressed BMP7 or exogenously administered rhBMP7 reduces onset and progression of experimental diabetic nephropathy in rodents (23, 27, 28). Mechanistic studies indicate that BMP7 reduces the nuclear activity of TGF--induced smad signals through the inhibitory smad6 downstream of smad5 (30, 34).
In the present studies, we tested the hypothesis that BMP7 is an endogenous differentiation and survival factor for podocytes in a diabetes-mimicking environment by preventing changes in the actin cytoskeleton and caspase-3 activation, and we examined some of the signals that are involed.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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(10 U/ml). For subsequent differentiation, cells were thermo-shifted to 37°C and incubated in interferon-free medium for 14 days.
BMP7 and BMP receptors.
Secreted BMP7 was examined in podocyte-conditioned media by double antibody sandwich ELISA using commercially available reagents (R&D Systems, Minneapolis, MN). Cells were incubated with serum-free media containing 0.1% BSA and either normal glucose (5 mM) or high glucose (25 mM) or TGF- (50 pM) for 3 days. As an additional but independent measure, BMP activity was examined in conditioned media in a cell-based assay as follows: C3H10-B12-BRE-Luc mouse embryonic mesenchymal cells stably transfected with the BMP-responsive element of Id-1 coupled to a luciferase reporter were used in this assay (kindly provided by D. Logeart-Avramoglou, Université de Paris, Paris, France). Cells in 96-well plates were incubated overnight with conditioned media in triplicate (100 µl/well). Luciferase activity in cell lysates was measured in a luminometer using commercially available reagents (Promega). The assay is sensitive to BMP7 but not specific, i.e., also measures BMP2 and BMP4 but not TGF- or VEGF activity (11).
The expression of the type I receptors Alk2, -3, and -6 in podocytes was assessed by rtPCR compared with whole kidney and liver mRNA that had been extracted from normal adult FVB/N mice (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD). RNA was reverse transcribed with the Omniscript RT kit (Qiagen, Hilden, Germany) and the PCR reactions were performed using specific primers (Table 1) and conditions that had been optimized.
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(50 pM) for 3 days. Cells were washed three times with PBS and lysed in 2x Laemmli buffer containing 50 mM DTT and heated to 100°C for 10 min. Proteins were electrophoretically separated in 10% SDS-PAGE gels (40 µg/lane) and electrotransferred to nitrocellulose. Blocked membranes were incubated with anti-Alk2 (Abgent, San Diego, CA), anti-Alk3, or anti-BMPRII (Santa Cruz Biotechnology, Santa Cruz, CA) for 16 h at 4°C, washed, and incubated with horseradish peroxidase-conjugated second antibody. Bands were visualized with enhanced chemiluminescence and captured on X-ray film.
BMP7-activated cell signals in podocytes.
Phosphorylation of smad1 and smad5 was determined by metabolic cell labeling and immunoprecipitation; phosphorylation of Erk1/2, JNK1/2, and p38 by BMP7 compared with TGF- was assessed by Western blot analysis with phospho-specific antibodies.
For the assessment of BMP7-induced smad phosphorylation, differentiated podocytes in six-well plates were washed 3x with phosphate/serum-free medium and then incubated with phosphate-free medium containing [32P]orthophosphate, 400 µCi/ml (MP Biomedicals, Irvine, CA) at 37°C for 1 h (1 ml/well). rhBMP7 (kind gift from Curis, Hopkinton, MA) was added to aliquot wells at a final concentration of 1 nM; control wells received equal amounts of solvent (1 mM acetic acid) and cells were incubated for 30 min at room temperature. Media were aspirated and cells were washed 2x with PBS and lysed in RIPA buffer containing protease inhibitor cocktail (Roche Diagnostic, Indianapolis, IN) and 20 mM Na-orthovanadate. Cells were disrupted by freeze-thawing and scraping. Lysates were precleared by centrifugation and cleared with protein-A/G agarose and then incubated with 4 µg/ml of anti-smad1 or anti-smad5 (Santa Cruz Biotechnology) for 1 h at 4°C on a rocker. After addition of 40 µl/ml of a 1:1 slurry of protein-A/G-conjugated agarose, incubations were continued for 4 h. Immunoprecipitates were collected by centrifugation, washed 4x with RIPA buffer, taken up with 2x reducing Laemmli buffer, electrophoresed in 10% SDS-PAGE gels, transferred to nitrocelulose, and autoradiographed. Membranes were subsequently Western blot analyzed with anti-smad 1 or anti-smad5, respectively.
For the assessment of nonsmad signals that were differentially induced in podocytes by BMP7 or TGF-, cells were incubated with rhTGF-1 (Biosource/Invitrogen, Camarillo, CA) or rhBMP7, each at 1 nM for 1 h; cells were washed 3x and lysed in 2x reducing Laemmli buffer. Western blot analysis was performed essentially as described above using antibodies against phospho-Erk1/2, phospho-, and total p38 (Cell Signaling Technology, Danvers, MA), total Erk1/2, phospho-, and total JNK1/2 (Santa Cruz Biotechnology).
Assessment of podocyte differentiation. To examine changes in cell shape and F-actin distribution, differentiated podocytes on collagen I-coated coverslips were incubated with high glucose or glycated albumin (400 µg/ml, Sigma) in media containing 0.1% FCS in the presence or absence of rhBMP7, 1 nM, for 3 days. Cells were then fixed and stained with Alexa Fluor 488-conjugated phalloidin (Molecular Probes, Eugene, OR).
The expression of several podocyte differentiation markers was examined in differentiated podocytes upon incubation with high glucose or TGF- by competitive rtPCR or Western blot analysis. Cells were incubated with normal glucose (5 mM) or high glucose (25 mM) or TGF- (100 pM) for 24 h. CD2AP, Neph1, and synaptopodin mRNA levels were measured by competitive rtPCR with specific primers (Table 1) and coamplification of 18 S rRNA in each tube as internal standard using commercially available reagents (Qiagen and Ambion, Austin, TX). Western blot analysis was performed as described above with anti-E-cadherin (BD Transduction Laboratories, San Jose, CA), and anti-podocin (Alpha Diagnostics, San Antonio, TX), and stripped membranes were reblotted with anti-gapdh (Fitzgerald Industries, Concord, MA) as loading control.
Cell survival, apoptosis, and caspase-3 activity.
To assess cell survival, podocytes were plated in collagen I-coated 96-well plates at 30,000 cells/well and allowed to differentiate at 37°C for 14 days. Cells were then incubated in RPMI 1640/0.1% FCS containing 5 mM glucose (control), rhTGF-1 (100 pM), rhTGF-1+rhBMP7 (1 nM), or high glucose (25 mM) with or without BMP7 (1 nM) for 3 days. Plates were frozen at –80°C and then thawed at room temperature and 200 µl/well of GR dye (1:400) in 1x lysis buffer (Molecular Probes) were added. Plates were shaken for 10 min at room temperature in the dark and fluorescence was measured at 520 nm at an excitation wavelength of 480 nm. Results are expressed as relative fluorescence units.
Apoptotic cells were identified among podocytes grown on collagen I-coated coverslips by in situ oligo ligation (ISOL). Cells were incubated with normal or high glucose or with normal glucose in the presence of TGF- (100 or 500 pM) for 24 h. As an osmolar control for the hyperosmolar challenge associated with the high glucose concentration, separate incubations with normal glucose ± mannitol, 20 mM, were also made. ISOL cytochemistry was performed using commercially available reagents and the manufacturer's procedure (Chemicon, Temecula, CA). Apoptotic cells were counted and expressed as a ratio of the total cell number.
Caspase-3 activity was measured in lysates from podocytes that were incubated for 24 h in six-well plates with normal glucose (5 mM, control), high glucose (25 mM), or TGF- (100 pM) with or without BMP7 (1 nM, n = 8), and incubations with normal glucose in the presence or absence of 20 mM mannitol as osmolar control were performed separately. Cells were then lysed in buffer containing 50 mM PIPES, 50 mM KCl, 5 mM EDTA, 2 mM MgCl2, and 1 mM DTT (pH 7.0). Cleared lysates were adjusted to a total protein concentration of 4 µg/µl. Assays were performed in 96-well plates containing 50 µl/well of cell lysates (or lysate buffer as blanks), and 2x assay buffer (250 mM HEPES, 1 mM EDTA, 50 mM KCl, 2 mM DTT, 0.01% CHAPS, pH 7.4), 50 µl/well was added. The mixture was shaken at room temperature for 5 min and 5 µl/well of the caspase-3-specific substrate, Ac-Asp-Gly-Val-Asp-pNA (Ac-DEVD-pNA, Bachem, Torrance, CA), in 5% NaHCO3 was added. Plates were mixed and incubated at 37°C for 6 h. Optical density was measured at 405 nm in a multiwell plate reader (Molecular Devices, Sunnyvale, CA). Results were expressed in percent of mean controls.
Requirement of smad5 for BMP7 effects on caspase-3 activation and actin distribution.
To assess whether smad5 is required (and sufficient) for the effects of BMP7 during high glucose or TGF--induced upregulation of caspase-3 activity or changes in actin distribution, differentiated podocytes were transfected with smad5 siRNA or a smad5 expression vector (or empty pCDNA3 vector as control). Highly efficient smad5 siRNA had been developed previously in this laboratory (30). Cells in six-well plates or on coverslips were transfected with smad5 siRNA at a final concentration of 25 nM using siPORT amine reagent (Ambion). Cells in other wells or coverslips were transfected with pcDNA3-smad5 (kindly provided by A. Moustakas and S. Souchelnytskyi, Uppsala, Sweden), 0.8 µg/well with Effectene reagent (Qiagen) at a ratio of 1:12.5 or empty vector (control). For caspase-3 measurement, cells were incubated for 24 h and media were exchanged to low serum (0.1%) media containing TGF- (100 pM) or glucose (25 mM) ± BMP7 (1 nM) for 24 h. After incubations, cells on coverslips were stained with Alexa Fluore 488-conjugated phalloidin for fluorescence microscopical assessment of F-actin distribution. Quantitative measurements of F-actin content were performed separately in identically treated cells that were grown and differentiated in collagen I-coated 12-well plates (n = 4 each). One group of cells treated with normal glucose + 20 mM mannitol was included as osmolar control. After the staining of the attached cells with conjugated phalloidin, cells were washed three times with buffer and the dye was extracted with ice-cold ethanol at –20°C. Cell debris was separated from the alcoholic dye extract by centrifugation and the concentration of the conjugated phalloidin was measured with a fluorometer (excitation 495 nm; emission 518 nm). Raw data were corrected for the cell number per well and for the cell protein content per well which were measured in parallel but separate wells by counting in a cytometer after detachment and with the DC protein assay (Bio-Rad, Hercules, CA), respectively.
Statistical analyses were performed with ANOVA and the Newman-Keuls multicomparison test. A probability of less than 5% (P < 0.05) was defined as significance of differences between group means.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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substantially reduces BMP7 secretion (Fig. 1A). Podocyte-derived BMP7 is bioactive, and its bioactivity also decreases upon exposure to high glucose (Fig. 1B). Although cell attrition due to apoptosis may contribute to the loss of BMP7, it clearly exceeds what would be expected purely by loss of cell number. Podocytes express the BMP type II receptor, BMPRII, and the BMP7-sensitive type I receptors Alk2 and Alk3 but not Alk6 (Fig. 1, C and D) consistent with the possibility of autocrine modes action of BMP7 in these cells. Alk3 mRNA levels were greater in podocytes compared with whole kidney extracts suggesting low levels of expression of this type I receptor in tubular cells compared with podocytes (Fig. 1C). Neither high glucose nor TGF- substantially affects the BMPRII or Alk2/3 levels (Fig. 1D).
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tends to moderately reduce Erk1/2 and JNK1/2 phosphorylation but also increases phospho-p38 levels similar to BMP7 (Fig. 2A). Coincubation with BMP7 and TGF- does not change the phosphorylation of Erk1/2, JNK1/2, or p38 suggesting that modifications of these pathways are not a mode of action of BMP7 in the rescue from TGF-- or glycemia-induced injury.
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High glucose changes podocyte phenotype. In their differentiated state, podocytes express F-actin in longitudinal "stress fibers" (Fig. 3A). Incubation with high glucose for 72 h induces a change in cell shape; cells tend to transform into a more rounded shape; many podocytes loose their close connections with neighboring cells but do not detach. This is associated with redistribution of F-actin filaments along the cell border and disappearance of the stress fibers. Coincubation with BMP7 in the presence of high glucose reduces this change in cell shape and in the actin cytoskeleton (Fig. 3A). Similar observations were made in podocytes that were incubated with glucose (5 mM) in the presence or absence of glycated albumin (400 µg/ml) instead of high glucose (data not shown).
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Smad5 mediates the podocyte phenotype rescue by BMP7. For further evaluation of the involvement of smad5 as the mediator of podocyte phenotype protection by BMP7, smad5 levels were increased in podocytes by transfection with the pcDNA3-Smad5 expression vector, or smad5 levels were reduced by transfection with smad5 siRNA. Even in the absence of exogenously added BMP7, overexpression of smad5 reduces the changes in actin distribution upon exposure to high glucose (Fig. 4A). During smad5 knockdown with specific siRNA, exogenously added BMP7 failed to rescue podocytes from changes in the actin cytoskeleton that are induced by high glucose (Fig. 4B).
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BMP7 improves cell survival.
High glucose as well as TGF- increase the rate of podocyte apoptosis three- to fourfold (Fig. 5A) and reduce cell survival (Fig. 5C) which is associated with an increase in caspase-3 activity (Fig. 5B). This suggests a role of this execution caspase in high glucose- and TGF--induced apoptosis and reduced survival. BMP7 reduces caspase-3 activity that is induced by high glucose or TGF- and partially rescues podocyte survival (Fig. 5, B and C). BMP7 may act through the smad5 pathway in preventing caspase-3 activation by TGF-. Differentiated podocytes were transfected with smad5 siRNA to achieve smad5 knockdown; or with pcDNA-smad5 to overexpress this BMP7 smad signal (Fig. 6A). Cells were then incubated with TGF- (100 pM) in the presence or absence of BMP7 (1 nM) for 24 h and caspase-3 activity was measured in cell lysates (Fig. 6B). TGF- raised caspase-3 activity which was blocked by BMP7 but enhanced by smad5 knockdown (Fig. 6B). Moreover, smad5 knockdown reduced the effects of BMP7. Overexpression of smad5 mimicked the effect of BMP7 on the prevention of TGF--induced caspase-3 activation and further promoted the effects of BMP7 in experiments where exogenous BMP7 was added to smad5 overexpressing cells. Incubation of podocytes with high glucose (25 mM) for 24 h also enhances caspase-3 activity (Fig. 6C) which is thought to be mediated by TGF- (10, 12, 19, 25). High glucose-induced caspase-3 activation is also ameliorated by BMP7 (Fig. 5B), and this is mimicked by increased expression of smad5 (Fig. 6C) indicating that smad5 is sufficient. Osmotic exposure per se does not raise caspase-3 activity as was examined by incubation with normal glucose in the presence and absence of 20 mM mannitol (normal glucose: 100 ± 11%; mannitol: 91 ± 9%). In concert, these findings show that BMP7 utilizes smad5 in its reduction of caspase-3 activation by TGF- or high glucose in podocytes.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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40% of patients which is the single most common cause for end-stage renal disease requiring renal replacement therapy. Diabetes is associated with progressive reduction in the renal expression of BMP7 and increasing levels of TGF- in renal glomeruli (31, 35). Exogenously administered rhBMP7 or endogenous overexpression of BMP7 in podocytes reduces onset and progression of diabetic nephropathy in rodents (13, 23, 27, 28). Evolving diabetic renal injury is also associated with early drop-out of podocytes due to increased apoptosis (16, 25, 28, 33). Increased glomerular levels of TGF- and hyperglycemia (at least in part through raising TGF-) appear to cause diabetic podocyte loss through apoptosis. Both TGF- as well as high glucose levels cause podocyte apoptosis through caspase-3 activation, in part, through activation of p38 MAPK in conjunction with a second proapoptotic pathway involving smad7 downstream of TGF- (19, 25). Peters and associates (17) recently showed that TGF--induced podocyte apoptosis is accelerated in the presence of IGFBP-3, whereas costimulation with BMP7 and IGFBP-3 reduces apoptosis.
The localization of BMP7 expression in vivo in the normal adult nephron has undergone limited study, and findings are not without controversy. Data from normal human kidney is rather scarce (18, 32). Wetzel and his associates (32) did not find immunoreactive BMP7 in glomeruli or proximal tubules but only in the distal nephron. These investigators were also unable to demonstrate BMP7 mRNA by PCR in isolated glomeruli from normal human kidney. Rudnicki and co-workers (18) also did not detect immunoreactive BMP7 in human glomeruli but described proximal tubular expression of BMP7 which was increased in proteinuria. In contrast, there is ample evidence for glomerular expression of BMP7 in vivo specifically and exclusively in podocytes in rats (2, 3) and in mice (9, 13, 22, 28). Moreover, several of these in vivo observations provide convincing evidence for autocrine and/or paracrine actions of podocyte BMP7 (3, 13, 28). Paracrine modes of action on podocytes may also occur through BMP7 that is heavily expressed in distal tubules which neighbor the glomerulus, but this is hypothetical and unproven. However, there is strong evidence for hormonal (endocrine) activity of BMP7 which may well include actions on podocytes. BMP7 is present in circulating blood and in vivo studies recently demonstrated bioactivity of endogenous BMP7 at distant targets by endocrine modes (24). Such systemic functions of (renal) BMP7 have recently been proposed by Davies and associates (5, 6) based on extrarenal findings in rodents with chronic renal disease, and this is further supported by observations that systemic administration of very small dosages of rhBMP7 in rodents can boost its physiological functions. Moreover, BMP7, similar to other peptides within the TGF- superfamily, avidly binds to extracellular matrix proteins, especially fibronectin and, hence, basement membranes could serve as a local reservoir (26). Thus endogenous BMP7 may act on target cells such as podocytes by autocrine, paracrine, and endocrine modes.
The podocytes in the present in vitro studies, similar to their in vivo counterparts at least in rodents, express BMP7 as well as BMPRII and the two type I receptors Alk2 and Alk3, which gives rise to autocrine in addition to para- or endocrine actions. With the present experiments, we tested the hypothesis that BMP7 is a phenotype maintenance and survival factor for podocytes during glycemic or TGF--induced injury.
Ambient high glucose levels cause several phenotype changes in cultured podocytes which include a rearrangement of the actin filaments (Fig. 3A). There is no evidence from the present studies that glycemic injury causes a major deviation from a general epithelial phenotype or, specifically, from the podocyte phenotype as indicated by the maintenance of E-cadherin levels and the levels of several podocyte markers (podocin, CD2AP, Neph1, synaptopodin). This correlates with findings in diabetic patients where levels of CD2AP and podocin are also similar compared with normal controls (1).
High glucose levels (as well as TGF-) raise the rate of podocyte apoptosis about fourfold and reduce cell survival (Fig. 5). The high glucose-induced changes in the structure of the actin cytoskeleton and caspase-3 activation leading to increased rates of apoptosis of podocytes are ameliorated by BMP7 which thereby improves cell survival in vitro.
Both high glucose and TGF- have previously been shown to induce apoptosis in podocytes (4, 10, 12, 14, 17, 19, 25). In most settings, activation of p38 and caspase-3 is required (4, 14, 19, 25). TGF--induced apoptosis may be independent of smad2/3 activation, but induction of smad7 may either amplify (19) or be required (14). The mechanisms of caspase-3 activation by high glucose or TGF- are thought to involve oxidant-derived injury (10, 25).
A specific question that is addressed in the present studies is how BMP7 reduces caspase-3 activation. It could have been possible that BMP7 reduces p38 phosphorylation, although TGF- increases p-p38 levels, so did BMP7 and coincubation with both peptides does not reduce p-p38 (Fig. 2). The effects of BMP7 on reducing caspase-3 activation cannot be mediated by Erk1/2 because BMP7 and TGF- both lower pErk1/2 but these two cytokines have disparate effects on caspase activity.
In podocytes, BMP7 efficiently phosphorylates smad5 (Fig. 2B). We previously showed that smad5 is also the preferred R-smad for BMP7 in mesangial cells (30). As shown in the present studies, smad5 knockdown prevents the effects of BMP7 on high glucose-induced changes in actin distribution in podocytes and overexpression of smad5 mimics, at least to some extent, the effects of exogenously added BMP7. Moreover, smad5 knockdown reduces the effects of BMP7 on TGF--induced caspase-3 activation, whereas forced expression of smad5 acts similar to BMP7 and reduces TGF-- or high glucose-induced caspase-3 activation. Thus smad5 is required and sufficient and mediates these effects of BMP7.
In summary, these in vitro studies provide mechanistic evidence for a role of autocrine, paracrine, and/or endocrine BMP7 and its signaling protein, smad5, as a podocyte differentiation and survival factor. Reduction of BMP7 early in the evolution of diabetic nephropathy contributes to podocyte injury and loss.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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1 potentiates renal tubular epithelial cell death by a mechanism independent of Smad signaling. J Biol Chem 278: 12537–12545, 2003.-dependent fibrogenesis in mesangial cells. Am J Physiol Renal Physiol 284: F1006–F1013, 2003.1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med 9: 964–968, 2003.[CrossRef][Web of Science][Medline]This article has been cited by other articles:
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  L. De Rosa, D. Antonini, G. Ferone, M. T. Russo, P. B. Yu, R. Han, and C. Missero p63 Suppresses Non-epidermal Lineage Markers in a Bone Morphogenetic Protein-dependent Manner via Repression of Smad7 J. Biol. Chem., October 30, 2009; 284(44): 30574 - 30582. [Abstract] [Full Text] [PDF]
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 T. Turk, J. W. Leeuwis, J. Gray, S. V. Torti, K. M. Lyons, T. Q. Nguyen, and R. Goldschmeding BMP Signaling and Podocyte Markers Are Decreased in Human Diabetic Nephropathy in Association With CTGF Overexpression J. Histochem. Cytochem., July 1, 2009; 57(7): 623 - 631. [Abstract] [Full Text] [PDF]
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 T. Q. Nguyen, P. Roestenberg, F. A. van Nieuwenhoven, N. Bovenschen, Z. Li, L. Xu, N. Oliver, J. Aten, J. A. Joles, C. Vial, et al. CTGF Inhibits BMP-7 Signaling in Diabetic Nephropathy J. Am. Soc. Nephrol., November 1, 2008; 19(11): 2098 - 2107. [Abstract] [Full Text] [PDF]
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 R. V. Durvasula and S. J. Shankland Activation of a local renin angiotensin system in podocytes by glucose Am J Physiol Renal Physiol, April 1, 2008; 294(4): F830 - F839. [Abstract] [Full Text] [PDF]
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