Tubular Epithelial Cell Dedifferentiation Is Driven by the Helix-Loop-Helix Transcriptional Inhibitor Id1

Cell Culture and Treatment

Human proximal tubular epithelial cell line (HKC clone 8) was maintained as described previously (18). Serum-starved HKC-8 cells were treated with recombinant TGF-β1 (R&D Systems, Minneapolis, MN) for various periods of time at the concentration of 2 ng/ml except otherwise indicated. The cells then were collected for Northern and Western blot analyses and immunofluorescence staining. In some experiments, cells were pretreated for 30 min with various chemical inhibitors at the concentrations specified, followed by incubation with TGF-β1. PD98059, wortmannin, myristoylated protein kinase A inhibitor, and SC 68376 were purchased from Calbiochem (La Jolla, CA). Small-molecule ILK inhibitor KP392 was described elsewhere (19,20).

Affymetrix Microarray Analysis

A high-throughput gene expression profiling was carried out by using Affymetrix chip analysis (Affymetrix, Santa Clara, CA), as described previously (21,22). Preparation of cRNA, hybridization, and scanning of the arrays were performed essentially according to the protocols specified by the manufacturer. Briefly, double-stranded cDNA was generated from total RNA that were isolated from the HKC-8 cells by using a reverse transcription kit (Invitrogen, Carlsbad, CA) and T7-d(T)₂₄ primer. Biotin-labeled cRNA was prepared from the double-stranded cDNA by in vitro transcription using MEGAscript system (Ambion, Austin, TX) and fragmented at 95°C for 35 min. The fragmented cRNA then were hybridized with an Affymetrix human Genome U95Av2 Array chip (HG-95Av2) set that was composed of approximately 12,000 fully sequenced and characterized genes. After the hybridization cocktails were removed, the chips then were washed, stained with streptavidin phycoerythrin (Molecular Probe, Eugene, OR), incubated with biotinylated mouse anti-streptavidin IgG, and re-stained with streptavidin phycoerythrin. The chips were scanned in an HP ChipScanner (Affymetrix) to detect hybridization signals. Data were analyzed using GeneChip software (Affymetrix).

Northern Blot Analysis and Reverse Transcriptase–PCR

Total RNA was extracted from the cultured cells and kidney tissue by using TRIzol reagent (Invitrogen). Northern blot analysis for gene expression was carried out by the procedures described previously (23). Reverse transcriptase–PCR (RT-PCR) was performed by standard protocol as described elsewhere (24). The primer sequences for mouse Id1 were as follow: 5′-CATGAACGGCTGCTACTCAC (sense) and 5′-GTGGTCCCGACTTCAGACTC (antisense). The primers for mouse β-actin were described previously (24).

Western Blot Analysis

Western blot analysis was performed essentially according to an established procedure (18). The primary antibodies used were as follows: Anti-Id1 (sc-488), anti-HEB (Hela E-box binding factor; sc-357), and anti-actin (sc-1616; Santa Cruz Biotechnology, Santa Cruz, CA); anti-α-smooth muscle actin (α-SMA; clone 1A4; Sigma, St. Louis, MO), anti–E-cadherin (clone 36), and anti-fibronectin (clone 10; BD Transduction Laboratories, Lexington, KY); anti-ILK (05–575; Upstate Biotechnology, Charlottesville, VA); anti-Myc (2276; Cell Signaling Technology, Danvers, MA); and anti–glyceraldehyde-3-phosphate dehydrogenase (Ambion). Quantification was performed by measurement of the intensity of the bands with the use of NIH Image analysis software.

Construction of Id1 Expression Vector and Establishment of Stable Cell Line

Human full-length coding sequence of Id1 cDNA was cloned by using a standard RT-PCR protocol (25). The PCR product was cloned into the mammalian expression vector pcDNA3 (Invitrogen) and confirmed by DNA sequencing at the University of Pittsburgh Biomedical Research Support Facilities. Flag- or Myc-tagged Id1 expression vectors also were constructed by standard subcloning techniques and confirmed by sequencing. HKC-8 cells were transfected with Id1 expression vector using the Lipofectamine 2000 (Invitrogen). The empty vector pcDNA3 was used as a mock-transfection control. Neomycin-resistant clones were selected and expanded individually, as described previously (10), and ectopic expression of Id1 was confirmed by Western blot analysis. The stable cell lines that overexpressed SnoN (Ski-related novel gene, non–Alu-containing), Smad7, or ILK were established previously and described elsewhere (10,26).

Immunofluorescence Staining

Indirect immunofluorescence staining was performed using an established procedure (18). Briefly, cells that were cultured on coverslips and kidney cryosections were fixed with cold methanol:acetone (1:1) for 10 min at −20°C. After blocking with 20% normal donkey serum in PBS buffer, the slides were incubated with the primary antibodies against E-cadherin, fibronectin, Id1, and ZO-1 (61–7300; Invitrogen). For visualization of the primary antibodies, slides were stained with cyanine Cy2- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Kidney cryosections also were stained with renal proximal tubular marker fluorescein-conjugated lectin from Tetragonolobus purpureas (Sigma). Stained slides were viewed with a Nikon Eclipse E600 Epi-fluorescence microscope equipped with a digital camera (Melville, NY).

Plasmid Construction, Transfection, and Reporter Gene Assay

The E-cadherin promoter-luciferase reporter plasmid was provided by Dr. A. Garcia de Herreros (Universitat Pompeu Fabra, Barcelona, Spain) (27). The E-box SV-promoter reporters were constructed by insertion of oligonucleotides that contained either wild-type or mutant E-boxes in the front of SV-40 promoter (pGL3-promoter vector; Promega, Madison, WI). HKC-8 cells were co-transfected with reporter plasmids and an internal control reporter (pRL-TK; Promega). Luciferase assay was performed using the Dual Luciferase Assay System kit (Promega) (25). Relative luciferase activity (arbitrary unit) was reported after normalizing for transfection efficiency.

Immunoprecipitation

Immunoprecipitation experiments were performed using similar methods as described previously (28). Briefly, cell lysates were centrifuged at 12,000 × g for 10 min at 4°C. The resulting supernatants were collected for immunoprecipitation. After preclearing with normal host IgG, the lysates were immunoprecipitated overnight at 4°C with 2 μg of anti-Id1 or anti-HEB antibodies, followed by precipitation with 30 μl of protein A/G Plus-Agarose (Santa Cruz Biotechnology) for 3 h at 4°C. The precipitated complexes were immunoblotted with antibodies against HEB and Myc, respectively.

DNA Affinity Precipitation Assay

DNA–protein interaction was examined by a simple DNA affinity precipitation assay (25). Briefly, the 5′-biotinylated, double-stranded oligonucleotide (0.2 nM) that contained two tandem copies of E-box was mixed with 200 μg of whole-cell extracts for 3 h at 4°C. In some experiments, 100-fold excess of unlabeled oligonucleotides that contained either E-box or mutant E-box was included as competitor DNA in the reaction mixture. After streptavidin-agarose (10 μl of packed beads) was added, the incubation was continued for an additional 3 h at 4°C. The beads were washed, and the eluted proteins were analyzed by Western blotting with anti-HEB antibody.

Statistical Analyses

Unilateral ureteral obstruction (UUO) was performed in male CD-1 mice using an established procedure, as described previously (18). Statistical analysis of the data was performed using SigmaStat software (Jandel Scientific Software, San Rafael, CA).

Id1 Is Induced Rapidly during TGF-β1–Mediated EMT

We used a high-throughput gene expression microarray approach to identify the genes whose expression was altered in the initial stage of tubular EMT that was induced by TGF-β1. As shown in Figure 1A, there were 94 genes whose expression was altered by more than two-fold at 0.5 h in HKC-8 cells after TGF-β1 treatment. Of particular interest, Id1, a transcriptional antagonist that belongs to the HLH protein family (29,30), was induced markedly. RT-PCR studies confirmed the induction of Id1 mRNA in HKC-8 cells that were treated with TGF-β1 (data not shown).

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Figure 1.

The helix-loop-helix (HLH) transcriptional inhibitor of differentiation-1 (Id1) is induced rapidly during TGF-β1–mediated tubular epithelial-to-mesenchymal transition (EMT). (A) Microarray analysis revealed an early induction of Id1 in renal epithelial cells after TGF-β1 treatment. Human tubular epithelial cells (HKC-8) were incubated without or with 2 ng/ml TGF-β1 for 0.5 h, and total RNA was subjected to Affymetrix microarray analysis. (B) TGF-β1 induced Id1 mRNA expression in tubular epithelial cells. HKC-8 cells were treated with 2 ng/ml TGF-β1 for various periods of time as indicated. Total RNA was analyzed by Northern blot for Id1 expression. The same blot was stripped and reprobed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for ensuring equal loading of each lane. (C through F) TGF-β1 induced Id1 protein expression in a time- and dosage-dependent manner. HKC-8 cells were treated either with 2 ng/ml TGF-β1 for various periods of time (C and D) or various amounts of TGF-β1 for 1 h (E and F) as indicated. Whole-cell lysates were immunoblotted with antibodies against Id1 and actin, respectively. Graphic presentation of the relative abundances of Id1 (fold induction over the controls) after various treatments is shown (D and F). *P < 0.05 versus control; n = 3.

We then cloned and sequenced the entire coding region of human Id1 cDNA from HKC-8 cells. Figure 1B shows a Northern blot analysis of Id1 expression in tubular epithelial cells after TGF-β1 treatment. The steady-state level of Id1 mRNA in HKC-8 cells began to increase as early as 0.5 h, reached the peak between 1 and 3 h, and declined rapidly to baseline level at 6 h. Western blot analysis also revealed a rapid induction of Id1 protein expression after TGF-β1 stimulation. As shown in Figure 1, C and D, Id1 protein peaked at 1 h and declined at 6 h. The induction of Id1 protein by TGF-β1 also was dosage dependent (Figure 1, E and F).

Induction of Id1 Expression by TGF-β1 Depends on Smad Signaling

We next investigated the potential signaling pathways that lead to Id1 induction by TGF-β1 in HKC-8 cells. Previous studies revealed that TGF-β1 activates numerous signaling pathways, such as Smad2/3, p38 mitogen-activated protein kinase (MAPK), and Akt kinase (28). However, pharmacologic inhibition of p38 MAPK and Akt kinase by specific inhibitors did not affect Id1 induction by TGF-β1 (Figure 2A). Likewise, inhibition of extracellular signal–regulated kinase–MAPK or protein kinase A or C also did not influence TGF-β1–initiated Id1 induction (Figure 2A). To study the potential involvement of Smad signaling, we examined the Id1 inducibility in the tubular epithelial cells that overexpressed Smad co-repressor SnoN. As shown in Figure 2B, forced expression of SnoN largely abrogated Id1 induction by TGF-β1. Similarly, overexpression of inhibitory Smad7 significantly inhibited the TGF-β1–mediated Id1 induction (Figure 2, C and D). Therefore, Id1 induction by TGF-β1 depends on intracellular Smad signaling.

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Figure 2.

Id1 induction by TGF-β1 in renal epithelial cells depends on Smad signaling. (A) Pharmacologic inhibition of various signal transduction pathways did not affect Id1 induction by TGF-β1. HKC-8 cells were pretreated with various chemical inhibitors or vehicle (DMSO) for 30 min, followed by incubation in the absence or presence of 2 ng/ml TGF-β1 for 1 h. Specific inhibitors for phosphatidylinositol-3-kinase (10 nM wortmannin), Mek1 (10 μM PD98059), p38 mitogen-activated protein kinase (MAPK; 20 μM SC 68376), protein kinase A (PKA; 0.3 μM PKA inhibitor [PKAI]), and PKC (50 nM Ro-31-8220) were used. (B and C) Overexpression of either Smad transcriptional co-repressor SnoN (Ski-related novel gene, non–Alu-containing) or inhibitory Smad-7 inhibited Id1 induction by TGF-β1. Stable cell lines that overexpressed either SnoN (B) or inhibitory Smad-7 (C) were established by transfection of SnoN or Smad-7 expression vectors. Cell lines with mock transfection of empty pcDNA3 vector were used as controls. Cells were treated with 2 ng/ml TGF-β1 for various periods of time as indicated, followed by immunoblotting with antibodies against Id1 and actin, respectively. (D) Graphic presentation of relative Id1 abundance normalized to actin after TGF-β1 treatment in the Smad-7–overexpressing cells.

Forced Expression of Id1 Suppresses E-Cadherin and ZO-1 Expression

To investigate the functionality of Id1 induction in tubular epithelial cells, we examined the phenotypic changes after ectopic expression of exogenous Id1 by stable transfection. As shown in Figure 3A, four stable cell lines exhibited different levels of Id1 protein expression. Of note, the cell line with mock transfection of pcDNA3 vector displayed a similar level of Id1 as in the parental HKC-8 cells. It is interesting that ectopic expression of Id1 markedly suppressed epithelial E-cadherin. It seemed that the E-cadherin levels were correlated inversely with the abundance of Id1, suggesting a dosage-dependent effect of Id1 on E-cadherin suppression. Figure 3B shows a linear regression plot between Id1 and E-cadherin levels, with the correlation coefficient being 0.91.

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Figure 3.

Forced expression of Id1 suppresses E-cadherin and zonula occludens-1 (ZO-1) expression in tubular epithelial cells. Stable cell lines were established by transfection of Id1 expression vectors. Cell line with mock transfection of empty vector pcDNA3 (vector) was used as control. (A) Cell lysates were immunoblotted with antibodies against Id1, E-cadherin, and actin. Numbers (C1 through C4) indicate four individual clones that express various levels of Id1. (B) Linear regression showed a closely inverse relationship between Id1 and E-cadherin in tubular epithelial cells. The correlation coefficient (R²) is shown. (C and D) Immunofluorescence staining showed E-cadherin localization in HKC-8 cells that were transfected with empty vector (C) or pCMV-Id1 (D). (E and F) Forced expression of Id1 also suppressed ZO-1 expression in tubular epithelial cells. Immunofluorescence staining showed ZO-1 localization at the junctions of HKC-8 cells that were transfected with empty vector (E) or pCMV-Id1 (F). Bar = 5 μm.

We further examined E-cadherin expression in HKC-8 cells by an indirect immunofluorescence staining. As illustrated in Figure 3, C and D, compared with the controls, tubular epithelial cells with overexpression of Id1 lost E-cadherin staining in the plasma membrane. Similarly, ectopic expression of Id1 suppressed ZO-1, a tight junction-associated protein, in tubular epithelial cells (Figure 3, E and F).

Downregulation of Id1 Blocks TGF-β1–Initiated E-Cadherin Suppression

To assess whether Id1 induction is necessary for TGF-β1–mediated E-cadherin suppression, we examined the E-cadherin expression after downregulation of Id1 by antisense strategy in HKC-8 cells. As shown in Figure 4A, antisense strategy resulted in a considerable inhibition of Id1 expression in the absence or presence of TGF-β1. Figure 4B shows the E-cadherin levels in the controls or TGF-β1–treated HKC-8 cells after transient transfection with either sense or antisense Id1 vectors. Transfection of antisense Id1 partially restored E-cadherin expression, which was repressed by TGF-β1 (Figure 4B, lane 4 versus 6). As expected, transfection of Id1 in sense orientation or incubation with TGF-β1 suppressed E-cadherin expression in HKC-8 cells (Figure 4B, lane 1 versus lane 2 or 4). Hence, Id1 seems to be required for mediating TGF-β1–initiated E-cadherin suppression in tubular epithelial cells.

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Figure 4.

Expression of antisense Id1 largely blocks TGF-β1–mediated E-cadherin suppression. HKC-8 cells that were transfected with empty vector (vector), Id1 (pCMV-Id1), or antisense Id1 (pCMV-Id1-as) were incubated without or with 2 ng/ml TGF-β1. (A) Antisense inhibition of Id1 expression in the absence or presence of TGF-β1 was confirmed by Western blot analyses. (B) Ectopic expression of antisense Id1 largely abolished TGF-β1–initiated E-cadherin suppression in tubular epithelial cells.

Id1 Suppresses E-Cadherin Promoter by Repressing E-Box–Mediated Gene Transcription

To understand how Id1 mediates the suppression of E-cadherin, we studied its effect on E-cadherin promoter activity. As shown in Figure 5A, Id1 suppressed E-cadherin promoter activity in a dosage-dependent manner. Because E-cadherin promoter harbors three E-boxes, the cis-acting element that is capable of binding bHLH proteins and activating gene transcription, we reasoned that the E-boxes may mediate the transcriptional repression by Id1. To test it, we constructed two copies of E-boxes into the heterologous SV-40 promoter (Figure 5B). After transfection of this construct into HKC-8 cells, luciferase activity was suppressed by Id1 in a dosage-dependent manner (Figure 5C). However, Id1 did not affect luciferase activity when the E-box was mutated. These data suggest that the E-cadherin suppression by Id1 likely is mediated through modulating the E-box–controlled gene transcription.

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Figure 5.

Id1 suppresses E-cadherin promoter activity by repressing E-boxes–mediated gene transcription in tubular epithelial cells. (A) HKC-8 cells were transiently co-transfected with the reporter construct (pE-cad-luc) that contained E-cadherin promoter region that was linked to firefly luciferase reporter gene and increasing amounts of pCMV-Id1 as indicated. The relative luciferase activity (with control group = 1.0) was presented after normalization of transfection efficiency. Data are means ± SEM from three independent experiments. *P < 0.01 versus controls. (B and C) Id1 suppressed the transcriptional activity of the heterogeneous promoter containing E-box in tubular epithelial cells. Diagram depicts the construction of reporter vectors in which wild-type or mutant E-boxes were cloned into the SV-40 promoter luciferase reporter plasmid (B). The relative luciferase activity (with control group = 1.0) was presented after transfection with various reporter vectors. Data are means ± SEM from three independent experiments. *P < 0.01 versus controls.

Id1 Antagonizes the Trans-activating Action of HEB by Forming HEB/Id1 Heterodimeric Complex

To delineate further the underlying mechanism of Id1 suppression of E-cadherin, we examined the interaction between Id1 and the E-proteins, members of the bHLH protein family. Preliminary studies showed that the predominant form of bHLH proteins in tubular epithelial cells was HEB (data not shown). However, incubation of HKC-8 cells with TGF-β1 did not alter HEB expression (Figure 6A).

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Figure 6.

Id1 prevents basic HLH transcription factor HEB binding to the E-box by sequestrating its activity via physical interaction. (A) TGF-β1 did not affect HEB protein abundance in tubular epithelial cells. HKC-8 cells were treated with 2 ng/ml TGF-β1 for various periods of time as indicated. Cell lysates were immunoblotted with antibodies against HEB, Id1, and actin. (B) Co-immunoprecipitation revealed a physical association between HEB and Id1 in tubular epithelial cells. Cell lysates that were prepared from HKC-8 cells that were incubated with 2 ng/ml TGF-β1 were immunoprecipitated with antibody against Id1, followed by immunoblotting with anti-HEB as indicated. (C and D) Physical interaction of exogenous Id1 and HEB in tubular epithelial cells. HKC-8 cells were transfected with Myc-tagged Id1 expression vector, and cell lysates were immunoprecipitated with anti-Id1 (C) or anti-HEB (D). Normal IgG was used as a negative control. The precipitates then were immunoblotted with anti-HEB (C) or anti-Myc antibodies. (E) Biotinylated DNA precipitation assay showed a specific binding of HEB to the E-box in the E-cadherin promoter. HEB protein was detectable in the complexes that were precipitated by double-stranded biotinylated DNA that corresponded to the E-box in E-cadherin promoter. For competition experiments, a 100-fold E-box or mutant E-box also was included in the reaction mixture. The sequences of E-box and mutant E-box are shown at the bottom. (F) TGF-β1 reduced the binding of HEB to the E-box in tubular epithelial cells. HKC-8 cells were treated with 2 ng/ml TGF-β1 for various periods of time as indicated. Whole-cell lysates were subjected to biotinylated DNA precipitation assay. (G) Diagram shows the potential mechanism by which Id1 prevents E-protein from binding to the E-box via forming a heterodimeric, transcriptionally inactive complex.

We investigated the potential interaction between HEB and Id1 by co-immunoprecipitation. As shown in Figure 6B, HEB protein was detectable in the complexes that were precipitated by anti-Id1 antibody, suggesting a physical interaction between HEB and Id1. This interaction was increased after TGF-β1 treatment (Figure 6B). For confirmation of the authenticity of this interaction, Myc-tagged Id1 was expressed in HKC-8 cells by transient transfection, followed by immunoprecipitation with anti-Id1, anti-HEB, or normal IgG, respectively. As shown in Figure 6C, HEB was detected only in the complexes that were precipitated by anti-Id1 but not in that by control IgG. Likewise, Myc was found in the complexes that were precipitated by anti-HEB (Figure 6D). Similar results were obtained when Flag-tagged Id1 expression vector was used (data not shown). Figure 6E shows that HEB protein constitutively bound to the E-boxes in HKC-8 cells under basal condition, as demonstrated by DNA affinity precipitation assay. Addition of wild-type but not mutant E-box DNA completely abolished the binding (Figure 6E, lane 3), suggesting the specificity of this DNA–protein interaction. However, the binding of HEB to the E-box was reduced dramatically after TGF-β1 treatment (Figure 6F). Because TGF-β1 did not affect the abundance of HEB (Figure 6A), this suggests that increased Id1 binding to HEB protein would diminish the availability of HEB to the E-box, thereby sequestering its ability to trans-activate gene transcription (Figure 6G).

Overexpression of Id1 Is Not Sufficient to Confer a Complete EMT

Because Id1 overexpression leads to E-cadherin suppression, an early event in tubular EMT (6), we next examined whether increased Id1 results in mesenchymal transition of tubular epithelial cells. As shown in Figure 7, overexpression of Id1 failed to induce the de novo expression of α-SMA. Likewise, Id1 did not induce matrix metalloproteinase-2 (Figure 7B), a matrix-degrading enzyme that is of importance in disrupting tubular basement membrane. Finally, Id1 also failed to induce fibronectin expression (Figure 7C). Similar results were obtained when the cells were stained with anti-fibronectin antibody (Figure 7, D through G). As a positive control, TGF-β1 was capable of inducing all mesenchymal markers in HKC-8 cells, as previously reported (6). These results indicate that Id1 is unable to confer a mesenchymal transition of tubular epithelial cells.

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Figure 7.

Id1 overexpression does not confer mesenchymal transition of tubular epithelial cells. (A) Overexpression of Id1 did not induce α-smooth muscle actin (α-SMA) expression. Cell lysates that were prepared from the stable cell line that overexpressed Id1 (Id1-C1) or vector control were immunoblotted with antibodies against α-SMA and actin, respectively. HKC-8 cells that were treated without or with 2 ng/ml TGF-β1 for 48 h were used as controls. (B) Zymographic analyses showed that overexpression of Id1 did not induce matrix metalloproteinase-2 expression. (C) Overexpression of Id1 did not induce fibronectin expression. Cell lysates that were prepared from the stable cell line that overexpressed Id1 (Id1-C1) or vector control were immunoblotted with antibodies against fibronectin and actin, respectively. (D through G) Immunofluorescence staining showed fibronectin expression and localization after various treatments. (D) Control HKC-8 cells. (E) HKC-8 cells that were treated with TGF-β1. (F) Stable cell line with empty vector. (G) Stable cell line with Id1 overexpression (Id1-C1). Bar = 5 μm.

Id1 and ILK Are Independent Downstream Effectors of TGF-β1/Smad Signaling

Because overexpression of ILK also suppresses E-cadherin (10), we sought to investigate any potential relationship between ILK and Id1. As shown in Figure 8, TGF-β1 induced both ILK and Id1 expression in HKC-8 cells. However, overexpression of Id1 did not affect ILK expression (Figure 8A). Conversely, forced expression of ILK did not induce Id1 (Figure 8B). We found that selective inhibition of ILK activity by small-molecule inhibitor KP392 blocked TGF-β1–mediated fibronectin and α-SMA expression but failed to restore E-cadherin expression (Figure 8C). Further studies revealed that inhibition of ILK by KP392 did not abolish TGF-β1–induced Id1 expression (Figure 8D), which may be sufficient for E-cadherin suppression. Together, these data suggest that the loss of E-cadherin after TGF-β1 treatment is mediated by both Id1 and ILK. Because ILK but not Id1 induces mesenchymal transition (10), it seems that Id1 and ILK are two separate and parallel signaling mediators of TGF-β1/Smad that play divergent roles in tubular EMT (Figure 8E).

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Figure 8.

Id1 and integrin-linked kinase (ILK) are independent downstream effectors of TGF-β1/Smad signaling. (A) Id1 did not affect ILK expression in tubular epithelial cells. Cell lysates prepared from the stable cell line that overexpressed Id1 (Id1-C1) or vector control were immunoblotted with antibodies against ILK and actin, respectively. HKC-8 cells treated without or with 2 ng/ml TGF-β1 for 48 h were used as controls. (B) ILK did not induce Id1 expression in tubular epithelial cells. Cell lysates prepared from the stable cell line that overexpressed ILK (wt-ILK) or vector control were immunoblotted with antibodies against Id1 and actin, respectively. (C) Inhibition of ILK activity by small-molecule inhibitor KP392 blocked TGF-β1–mediated fibronectin and α-SMA expression but did not restore E-cadherin expression. HKC-8 cells were treated with TGF-β1 in the absence or presence of KP392 (50 μM) for various periods of time as indicated, and cell lysates were immunoblotted with antibodies against fibronectin, α-SMA, E-cadherin, and actin, respectively. (D) Inhibition of ILK did not abolish TGF-β1–induced Id1 expression. HKC-8 cells were pretreated with KP392 (50 μM) for 30 min, followed by incubation with or without TGF-β1 (2 ng/ml) for 1 h. Cell lysates were immunoblotted with antibodies against Id1 and actin, respectively. (E) Diagram depicts that Id1 and ILK are two independent, parallel signaling mediators of TGF-β1/Smad that play divergent roles in tubular EMT.

Tubule-Specific Induction of Id1 in the Fibrotic Kidney

In an effort to address the relevance of Id1 induction to tubular epithelial dedifferentiation in vivo, we investigated the expression of Id1 in the fibrotic kidney that was induced by UUO. Similar to in vitro situation, Id1 mRNA was induced rapidly and transiently in the obstructed kidney as early as day 1 after UUO (Figure 9, A and B). Id1 protein was increased in the obstructed kidney at day 1 after surgery and sustained to day 14 (Figure 9, C and D). We also examined the localization of Id1 protein in the obstructed kidney by immunofluorescence staining. As shown in Figure 9E, little or no Id1 staining was observed in sham-operated, normal kidney. However, strong Id1 staining was visible in the tubular epithelium of the obstructed kidneys (Figure 9H). Double staining for Id1 (red) and proximal tubular marker (green) revealed that Id1 was localized primarily in the proximal tubules in the obstructed kidney (Figure 9J). Of particular interest, Id1 was increased specifically in the degenerated, dilated tubules with an enlarged lumen (Figure 9J, asterisk). In morphologically intact proximal tubules, Id1 essentially was absent in the obstructed kidney (Figure 9J, arrow). These observations strongly suggest that Id1 induction is correlated closely with the dedifferentiation and degeneration of the tubular epithelium in vivo.

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Figure 9.

Id1 is induced specifically in the degenerated tubular epithelia of the fibrotic kidney. (A and B) RT-PCR analyses showed an early induction of Id1 mRNA in the fibrotic kidney that was induced by unilateral ureteral obstruction (UUO). RNA samples from three individual animals per time point (n = 3) were assessed for the mRNA expression of Id1 and β-actin, respectively. Relative Id1 mRNA levels (fold induction over the sham control) after correction with β-actin are presented (B). *P < 0.05 versus sham control. (C and D) Western blot analyses revealed an early and sustained induction of Id1 protein in the fibrotic kidney that was induced by UUO. Kidney homogenates were immunoblotted with antibodies against Id1 and actin, respectively. Samples from two representative animals were used at each time point. Relative Id1 protein levels (fold induction over the sham control) after correction with actin are presented (D). (E through J) Immunofluorescence staining displayed Id1 (red) and tubular cell marker lectin (green) localization in the sham kidney (E through G) and obstructed kidneys for 7 d (H through J), respectively. *Id1-positive tubules; arrows indicate renal tubules with negative Id1 staining. Bar = 15 μm.