Chordin-like 1 and twisted gastrulation 1 regulate BMP signaling following kidney injury - PubMed (original) (raw)

Chordin-like 1 and twisted gastrulation 1 regulate BMP signaling following kidney injury

Barry W Larman et al. J Am Soc Nephrol. 2009 May.

Abstract

Stimulation of the bone morphogenetic protein (BMP) pathway protects the kidney from acute and chronic injury. Numerous regulators in the kidney control BMP signaling, offering many targets for therapeutic manipulation. Here, we screened for modulators of BMP signaling in the ischemia-sensitive S3 segment and found that Chordin-like 1 is expressed in this segment of both the mouse and human nephron. Chordin-like 1 specifically antagonizes BMP7, which is expressed in the neighboring distal nephron, and this depends on the presence of the protein Twisted gastrulation. Upon ischemia-induced degeneration of the S3 segment, we observed a reduction in Chordin-like 1 expression coincident with intense BMP signaling in tubules of the recovering kidney. Restored expression accompanied proximal tubule epithelia redifferentiation, again coincident with decreased BMP signaling. We propose that Chordin-like 1 reduces BMP7 signaling in healthy proximal tubules, and the loss of this activity upon sloughing of injured epithelia promotes BMP7 signaling in repopulating, dedifferentiated epithelia. As regenerating epithelia differentiate, Chordin-like 1 is again expressed, antagonizing BMP7. These data suggest a mechanism for dynamic regulation of renoprotective BMP7 signaling in the S3 segment of the proximal tubule.

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Figures

Figure 1.

IRI reduces Chrdl1 expression. (A) Periodic acid-Schiff (PAS) staining shows tubule injury in cortex (top) and outer medulla (middle) after 45 min of renal ischemia at 24 h after surgery and after 20 d, when a substantial amount of tubule regeneration has taken place. The majority of tubular degeneration takes place in the outer medulla. In the cortex and outer medulla of the sham-operated control, proximal tubules show strong lumenal PAS staining (arrows), whereas at 24 h after ischemia, degenerated tubules of the outer medulla are filled with protein casts (*). After 20 d, large numbers of tubules with luminal PAS staining can be seen in the recovering kidney (arrows). This is reflected by staining with the proximal tubule marker lotus lectin in the outer medulla (bottom): Numerous proximal tubules display lumenal staining in the sham and 20 d after ischemia (arrows), whereas only trapping in protein casts of degenerated tubules (*) can be seen 24 h after ischemia. (B) Injury and recovery is reflected by dynamic expression of the injury marker Havcr1, the mouse ortholog of human KIM1. QPCR for Havcr1 was normalized to β-actin expression, and all assays were performed in triplicate. (C) RT-PCR screen of BMP antagonists expressed in the kidney 24 h after induction of ischemic injury indicates that levels of Chrdl1 expression are uniquely reduced. (D) RT-PCR showing dynamic expression of Chrdl1 compared with Chordin (inset) and QPCR of Chrdl1 expression (normalized to β-actin), both during a 20-d period after ischemic injury (n = 3). At 24 h after ischemia, expression of Chrdl1 is reduced to <5% of the level detected in the sham sample. The cDNA used in C and D (inset) was generated using RNA pooled from three animals. Loss of Chrdl1 expression comparable to that seen in the assay of pooled RNA was confirmed on five individual samples from injured kidneys (data not shown).

Figure 2.

Chrdl1 is expressed in the medullary proximal tubule of the nephron. (A) RT-PCR for Chrdl1 was performed on cDNA from cells representing distal tubule (MDCK), proximal tubule (HK-2), and collecting duct (mIMCD-3). Only the HK-2 proximal tubule cell line expresses Chrdl1. The PCR primers were designed to amplify within a single exon, allowing genomic DNA to serve as a positive control. The template for the negative control was synthesized using DNAsed RNA in a reverse transcription reaction in the absence of reverse transcriptase (No RT). (B) Chrdl1 is shown by in situ hybridization to be expressed in the outer medulla (M) but not in the papilla (P) or the cortex (C) (first panel); boxed region enlarged in the second panel. This expression pattern overlaps with the expression of the proximal tubule marker Lotus Tetragonolobus lectin but not with markers for distal tubule (E-cadherin), collecting duct (Dolichos Bifloris Agglutinin lectin [DBA]), or thick ascending limb (Tamm-Horsfall antigen). (C) Consistent with quantitative PCR data (Figure 1D), in situ hybridization for Chrdl1 shows that expression is reduced 24 h after ischemic injury and returns to approximately normal levels by 20 d after injury. The high-magnification insets show signal in tubular epithelial cells in the sham and at 20 d after ischemia, whereas at 24 h after ischemia, background signal results from nonspecific binding to cellular debris in lumenal protein casts (inset and Supplemental Figure S1). A control tested with a sense probe showed no specific staining (first panel).

Figure 3.

CHRDL1 protein is expressed in proximal tubules of the human renal medulla and is secreted by proximal tubule cells. (A) Proximal tubules are identified in paraffin sections of the human renal cortex (top) and outer medulla (bottom) by staining with lotus lectin (green). Co-staining with a polyclonal antiserum specific for CHRDL1 (red) in the left panels demonstrates co-localization with lotus lectin in tubules of the medullary region of the human kidney. CHRDL1 can also be seen, however, in some cortical convoluted tubules, possibly representing secreted protein or a protein expression pattern somewhat different from mouse. The right panels show negative control sections treated in an identical manner except for the omission of the CHRDL1 antibody. (B) HK-2 proximal tubule cells secrete CHRDL1. A goat polyclonal antiserum specific for CHRDL1 was used to precipitate protein from HK-2 conditioned medium (HK-2). Precipitated protein was immunodetected using a mouse mAb specific for CHRDL1, revealing a protein of approximately 60 kD. Although the predicted molecular weight of CHRDL1 is approximately 51 kD, previous immunoblotting studies with the recombinant protein have shown the molecular weight to be approximately 60 kD (data not shown), possibly as a result of extensive posttranslational processing. To control for the specificity of the assay and to predict the molecular weight accurately, HEK293 cells transfected with a control vector or a CHRDL1 expression vector were immunoprecipitated using the same conditions as HK-2 cells and immunoblotted on the same filter. The negative control (HEK293−) shows that the antibody does not precipitate confounding protein species, whereas the positive control (HEK293+) demonstrates that the approximately 60-kD band is indeed CHRDL1.

Figure 4.

CHRDL1 functions as a general BMP-signaling amplifier but specifically antagonizes BMP7 in the presence of TWSG1. (A) P19 embryonal carcinoma cells transfected with the BMP transcriptional reporter pBRE-Luc were incubated overnight with BMP4 (5 ng/ml) or BMP7 (10 ng/ml) and increasing amounts of CHRDL1 (50 to 400 ng/ml) or Chordin (100 to 800 ng/ml). Both CHRDL1 and Chordin amplify BMP4 signaling; however, in contrast to Chordin, which antagonizes BMP7 signaling, CHRDL1 amplifies BMP7 signaling in a dosage-responsive manner. (B) In the presence of TWSG1 (100 to 400 ng/ml), CHRDL1 (200 ng/ml) continues to act as an amplifier of BMP4 signaling unlike Chordin (400 ng/ml), which becomes a potent BMP4 antagonist. In contrast, CHRDL1 amplification of BMP7 signaling is converted to antagonism by addition of TWSG1 in a dosage-dependent manner. (C) In the P19 pBRE-luc reporter assay, the addition of TWSG1, CHRDL1, or Chordin (400 ng/ml) alone does not affect transcriptional activation. (D) CHRDL1 binds BMP7 and TWSG1 only in a trimolecular complex. A co-immunoprecipitation experiment in which CHRDL1 (500 ng/ml) is incubated in the presence of TWSG1 (500 ng/ml) and/or BMP7 (250 ng/ml) demonstrates that CHRDL1 binds TWSG1 and BMP7 only when all three proteins are present. No evidence of binding is detected when TWSG1 or BMP7 alone is incubated with CHRDL1. Recombinant proteins were immunoprecipitated with a goat polyclonal antibody against CHRDL1, and blots of goat IgG serve as loading controls. The last panel, a CHRDL1 immunoblot of an identical immunoprecipitation substituting an irrelevant goat antibody, demonstrates the absence of nonspecific binding by CHRDL1 to either the goat antibody or Protein G beads used in the experiment. The first lane of all blots contains recombinant protein as a positive control. (E) TWSG1 is strongly expressed in tubule epithelia of the mouse and human kidney. (Top) Strong immunohistochemical staining for TWSG1 in tubule epithelia of adult mouse kidney but weak staining in the glomerulus (G) and interstitial cell population (inset). (Right) Negative control using an antibody of the same species specific for macrophage. (Bottom) Immunofluorescent micrographs of an adult human kidney showing TWSG1 expression (red) in both proximal tubules (PT), marked green with Lotus lectin, and other tubules. As in the mouse, glomerular expression of TWSG1 is much weaker than seen in tubular epithelia. (Right) Negative control for TWSG1.

Figure 5.

CHRDL1 reduces BMP7-stimulated Smad activation and ID gene expression in the presence of TWSG1 but has no effect on BMP4 signaling. (A and B) In the presence of TWSG1, CHRDL1 inhibits Smad phosphorylation by BMP7 but not by BMP4. P19 cells were serum starved for 2 h and then incubated with BMPs (10 ng/ml) and antagonists for 2 h before lysis and Western blotting. Blots were probed for phosphorylated Smads 1, 5, and 8 and β-tubulin. In lanes 3, 4, and 5, Noggin (200 ng/ml), TWSG1, and CHRDL1 alone (400 ng/ml) were added. In lanes 6 through 9 CHRDL1 (400 ng/ml) was added together with increasing concentrations of TWSG1 (50, 100, 200, and 400 ng/ml). BMPs were incubated at 37 °C for 1 h with or without antagonists before application. Note that the arrow points to the band corresponding to pSmad1/5/8. The top band (arrowhead) is a contaminating band specific to P19 lysates and is not present in lysates from MDCK cells (compare C and D). (C and D) The effects of CHRDL1 and TWSG1 on BMP signaling can be reproduced in the MDCK kidney cell line. (E) RT-PCR showing expression of ID1, 2, and 3 genes in HK-2 cells stimulated with BMP4 or 7 (25 ng/ml) for 6 h in the presence of varying concentrations (100 to 400 ng/ml) of CHRDL1 and/or TWSG1. ID expression by HK-2 cells in response to BMP4 is unaffected by the presence of TWSG1. In cells incubated with BMP7, the addition of TWSG1 reduces ID gene expression.

Figure 6.

Overexpression of mouse Chrdl1 in the collecting duct substantially reduces BMP signaling in vivo. (A) Diagram of mouse Chrdl1 transgene construct driven by a collecting duct–specific enhancer element from intron 1 of the Bmp7 gene; quantitative PCR assay shows Chrdl1 overexpression in kidneys of two embryonic day 17.5 transgenic embryos (150 and 151) compared with wild-type. (B through D) Immunolocalization of phosphorylated Smads (red) in the embryonic day 17.5 wild-type kidney (B), and kidneys from transgenic embryos 150 and 151 (C and D) show that BMP signaling is substantially reduced by expression of the Chrdl1 transgene. Collecting ducts (CD) were localized by staining with the lectin Dolichos Biflorus Agglutinin (green), and nuclei were counterstained with DAPI (blue).

Figure 7.

BMP signaling in the regenerating postischemic kidney is dynamic. (A) BMP signaling visualized by immunofluorescent staining for pSmad1/5/8 (red) shows the increase in signaling associated with recovery from ischemic injury. Proximal tubules are marked with lotus lectin (green) and nuclei with DAPI (blue). In the sham control, limited nuclear pSmad1/5/8 is seen in the cortex, and none can be seen in the outer medulla. After injury, nuclear pSmad1/5/8 can be seen in degenerating proximal tubules of the outer medulla. Extensive BMP signaling is maintained at 7 and 14 d after ischemia, at which point the majority of proximal tubule cells have been sloughed off and are being replaced. pSmad1/5/8 staining is prominent in cells repopulating proximal tubules of the outer medulla (arrows). At 20 d after injury, the level of signaling approaches baseline in the cortex and is again undetectable in the medulla. The sensitivity of the pSmad1/5/8 antibody in immunofluorescence is lower than in immunohistochemical assays, allowing clearer visualization of changes in signaling. (Supplemental Figure S3). (B) In homeostasis, BMP7 ligand emanating from the distal tubule and thick ascending limb is antagonized in the S3 segment of the proximal tubule by CHRDL1 in conjunction with Twisted gastrulation. In ischemic injury, the epithelium of the S3 segment is particularly prone to injury and death. As a result, CHRDL1 expression is reduced, with a concomitant reduction in the antagonism of BMP7 signaling. The resulting increase in BMP signaling is part of the regenerative process that rebuilds the tubule epithelia. As the cells of the recovering epithelia fully redifferentiate, a state of homeostasis is again achieved with BMP7 signaling antagonized by the presence of CHRDL1 and Twisted gastrulation.

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Chordin-like 1 and twisted gastrulation 1 regulate BMP signaling following kidney injury - PubMed (original) (raw)