Wnt-Dependent β-Catenin Signaling Is Activated after Unilateral Ureteral Obstruction, and Recombinant Secreted Frizzled-Related Protein 4 Alters the Progression of Renal Fibrosis : Journal of the American Society of Nephrology

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Pathophysiology of Renal Disease and Progression

Wnt-Dependent β-Catenin Signaling Is Activated after Unilateral Ureteral Obstruction, and Recombinant Secreted Frizzled-Related Protein 4 Alters the Progression of Renal Fibrosis

Surendran, Kameswaran*; Schiavi, Susan; Hruska, Keith A.*

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Journal of the American Society of Nephrology 16(8):p 2373-2384, August 2005. | DOI: 10.1681/ASN.2004110949
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Wnt4 expression is increased after renal epithelial injury in different rodent models of tubulointerstitial fibrosis and can disrupt the renal tubular epithelial structures (13). Wnt genes code for secreted proteins that associate with the cell surface and are capable of signaling via members of a family of receptors named frizzled (4). The frizzleds consist of an extracellular cysteine-rich domain (CRD), seven transmembrane regions, and a C-terminal intracellular domain (5). One signaling pathway downstream of Wnt that interacts with the CRD of frizzled receptors is mediated via stabilization of β-catenin (4), which then translocates into the nucleus and interacts with the T cell factor (TCF)/lymphoid enhancing factor (LEF) family of DNA binding proteins to alter gene expression (68). Because fibronectin, a marker of fibrosis, is upregulated by β-catenin and TCF/LEF (9) and its deposition has been correlated with increased Wnt4 expression after renal injury (2), we wanted to analyze the effects of an inhibitor of the Wnt-mediated β-catenin signaling on the progression of renal fibrosis.

A gene family related to the frizzled family of Wnt receptors codes for secreted proteins that lack the seven transmembrane-spanning regions of the frizzled proteins. These secreted frizzled-related proteins (sFRP) have a cysteine-rich domain that can bind Wnt proteins and frizzled receptors and may perturb the Wnt-frizzled interaction to modulate the functions of Wnt proteins (1012). Five murine sFRP genes have been identified, but their expression and function during the progression of renal fibrosis are unknown. Another secreted family of Wnt signaling inhibitors/modulators is coded for by the Dickkopf (dkk) family of genes. The expression and function of the four identified dkk genes during the progression of renal tubulointerstitial fibrosis are also unknown. The Dkk, at least Dkk1, can inhibit Wnt signaling that is dependent on the Wnt co-receptors LDL receptor–related proteins 5 and 6 (LRP5/6). Dkk1 can interact with the Wnt co-receptor LRP6 and cause the endocytosis of LRP6 to downregulate Wnt signaling (1315).

To identify Wnt signaling functions and to determine the possibility of inhibiting Wnt signaling during progression of renal tubulointerstitial fibrosis, we determined the expression patterns of sFRP and dkk genes. In a model of renal fibrosis, induced by unilateral ureteral obstruction (UUO), the expression of dkk1 and sFRP4 was consistently increased. Here we report our findings on the expression pattern of sFRP4 during renal tubulointerstitial fibrosis and test the ability of recombinant sFRP4 to alter the progression of renal fibrosis.

Materials and Methods

UUO was performed on anesthetized FVB/N (Taconic, Germantown, NY), C57BL/6J (Jackson Laboratories, Bar Harbor, ME), and Tg(GFPU)5Nagy/J (Jackson Laboratories) strains of mice. Mice were 6 to 11 wk of age when UUO was achieved by surgical cautery of the left ureter approximately 15 mm from the renal pelvis. Approval was obtained from the Washington University Institutional Animal Care and Use Committee for all experiments involving animals. Kidneys were harvested 1 to 28 d after obstruction. Uninjured kidneys were harvested from 7- and 11-wk-old mice. Obstructed kidneys were frozen in OCT embedding medium for RNA in situ hybridization, snap-frozen in liquid nitrogen for RNA and protein extraction, or fixed in 10% neutral-buffered formalin for immunohistochemistry.

Reverse Transcription–PCR

Total kidney RNA was extracted using RNeasy Midi-kit (Qiagen, Studio City, CA). Reverse transcription (RT) was performed using 5 μg of RNA, 2.5 μg of oligo(dT)15, 50 U of AMV reverse transcriptase (Fisher Scientific, Pittsburgh, PA), 5 mM MgCl2, and 1 mM of each dNTP in RT buffer (Fisher), at 42°C for 1 h. One twenty-fifth of the total RT product volume was used in each PCR. Forward and reverse primers were from different exons. PCR was performed using KlentaqLA DNA polymerase (D5062; Sigma, St. Louis, MO) in 1× KLA buffer that contained 1.3 M betaine (B2754; Sigma), 50 μM cresol red (C9877; Sigma), 50 μM of each dNTP, and 20 pmol of each primer. The primers are listed in Table 1. PCR conditions were 93°C for 1 min and 22 to 35 cycles of 93°C for 20 s and 68°C for 3 min in a thermal cycler (Eppendorf).

RNA in situ hybridization was performed using 10- to 12-μm sections of OCT-embedded kidneys as described previously (2). The sFRP4 antisense riboprobe was transcribed using linearized sFRP4 partial cDNA template, [α-33P] UTP (Amersham Biosciences, Piscataway, NJ), and the Promega in vitro transcription system (Promega, Madison, WI) (2). The sFRP4 partial cDNA template was amplified by RT-PCR, and the RT-PCR product was ligated into the pZero2.0 plasmid (Invitrogen, San Diego, CA) and the sequence was verified.

Administration of Recombinant sFRP4

Mice received intraperitoneal injections of 0, 720 ng, or 7.2 μg of recombinant sFRP4 dissolved in a 200-μl volume of PBS + 0.1% BSA. The production of recombinant sFRP4 was described previously (16). The recombinant sFRP4 was injected into the intraperitoneal cavity in the vicinity of the obstructed kidney. The injections were given 2, 4, and 6 d after initiation of UUO. Obstructed kidneys were harvested 7 d after the initiation of UUO. For analysis of E-cadherin expression, mice received an injection of vehicle or 720 ng of sFRP4 every 24 h beginning at the time of obstruction and ending 4 d after obstruction. For determining the renal accumulation of recombinant sFRP4, mice received an injection of 7.2 μg of sFRP4 in the vicinity of their left kidneys. Twenty and 40 min after injection, the left kidneys were harvested and analyzed for the presence of recombinant sFRP4.

Western Blot Analysis

Whole kidneys were homogenized in 50 mM Tris (pH 7.4) buffer that contained 150 mM NaCl, 1% NP-40, 0.2 mM sodium orthovanadate, and protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany) using a polytron homogenizer. This SDS-deficient lysis buffer was used to minimize solubilization of membrane-associated proteins, to detect total cytosolic β-catenin levels without the membrane/E-cadherin–associated β-catenin. Lysates were incubated on ice for 30 min and centrifuged at 10,000 × g for 10 min at 4°C, and protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA). Equal protein amounts of each sample were denatured using β-mercaptoethanol in Laemmli buffer and separated by SDS-PAGE. Proteins were transferred to PVDF membranes (Immobilon-P; Millipore, Bedford, MA), and membranes were probed with anti-fibronectin (#60040606117; Research Diagnostics, Flanders, NJ), anti-cellular fibronectin (MAB1940; Chemicon, Temecula, CA), anti–α-smooth muscle actin (α-SMA; A2547; Sigma), anti–β-tubulin (T4026; Sigma), anti–active-β-catenin (05-665; Upstate Biotechnology, Lake Placid, NY), or anti–β-catenin (BD Biosciences, San Jose, CA) or anti–E-cadherin (BD Biosciences) antibodies. The anti–β-catenin antibody recognizes the C-terminal part of β-catenin and should detect β-catenin independent of whether β-catenin is phosphorylated or not at residues 33, 37, 41, or 45, located in the N-terminal region. A rabbit polyclonal antibody against a 14–amino acid peptide in the netrin domain of sFRP4 was used to detect sFRP4 (Genzyme, Cambridge, MA). At best, only four of the 14 amino acids in the peptide are conserved among the five sFRP proteins. In addition, this polyclonal antibody was tested for specificity and does not recognize recombinant mouse sFRP2 or human sFRP3. An antibody against the V5 epitope (Invitrogen) was used to detect the presence of recombinant sFRP4 in the obstructed kidneys. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) and supersignal west-pico-chemiluminescent kit (Pierce, Rockford, IL) were used to visualize the amount of each protein. Signal intensities from immunoblots were quantified using Image J software (National Institutes of Health, Bethesda, MD). For comparison of protein levels among kidney extracts run on different blots, signal intensities for each protein were normalized with β-tubulin intensities, and the average of the normalized signal intensities in the 7-d obstructed kidneys of mice that were treated with 0 μg of sFRP4 was set to 1 U of expression for each protein.

Immunohistochemistry was performed on 4-μm kidney sections. The endogenous peroxidase activity was quenched in 3% H2O2 for 10 min, followed by blocking for 30 min at room temperature with PBS that contained 1% BSA (A7906; Sigma), 2% normal donkey serum, and 2% donkey anti-mouse IgG Fab fragment (Jackson ImmunoResearch) and then incubation with anti–α-SMA (A2547; Sigma) or anti–β-catenin (Sigma) or anti-sFRP4 antibody, for approximately 14 h at 4°C. The sections were rinsed in PBS, incubated for 1 h at room temperature with HRP- or cy3-conjugated secondary antibodies (Jackson ImmunoResearch), and rinsed in PBS. The HRP was developed with a peroxidase substrate (SK-4100; Vector Laboratories, Burlingame, CA), before counterstaining with hematoxylin. For the fluorescence (cy3) detection, the tissue sections were incubated with Hoechst dye.

Counting Myofibroblasts

Five sections of each obstructed kidney were analyzed for α-SMA protein expression. Ten nonoverlapping digital images of ×40 magnification were taken per section using a Zeiss microscope. Each image was opened in Photoshop and overlaid with a 130-point grid. The number of points that coincided with α-SMA–positive (brown) interstitial cells was recorded as the number of myofibroblasts present in a ×40 field. In total, 50 images per obstructed kidney were analyzed. The average of the total number of myofibroblasts per obstructed kidney of vehicle-treated mice was set as 100 U for convenient comparison of vehicle- versus sFRP4-treated mice.

Real-time RT-PCR was done as described in the RT-PCR section, except that SYBR Green dye replaced cresol red, and PCR was performed using the Mx4000 multiplex quantitative PCR system (Stratagene, La Jolla, CA). The SYBR Green with Dissociation Curve platform was used for analysis of the data as described by the manufacturer. The standard curves were made using the cDNA synthesized by reverse transcription using mouse kidney RNA.

Statistical Analyses

SigmaPlot software was used to perform t tests for comparisons between the obstructed kidneys of mice that were treated with 0 μg of sFRP4 versus obstructed kidneys of mice that were treated with 7.2 μg of sFRP4.


Wnt-Dependent β-Catenin Signaling Increased along with Markers of Fibrosis after UUO

After UUO, cytosolic β-catenin is increased (Figure 1) and may reflect an increase in Wnt-dependent β-catenin signaling. We further confirmed increased Wnt signaling activity after UUO by measuring the amount of β-catenin that is not phosphorylated on both Ser37 and Thr41 (active β-catenin; Figure 1) (17). In the absence of Wnt signaling, β-catenin is phosphorylated on Ser33, Ser37, Thr41, and Ser45, which target β-catenin for ubiquitination and subsequent degradation. Wnt signaling inhibits GSK3β-mediated β-catenin phosphorylation on Ser33, Ser37, and Thr41 residues (18). Along with increased Wnt signaling, we confirmed that UUO induces an increase in total fibronectin, cellular fibronectin that contains the extra type IIIA region (19), and α-SMA (Figure 1).

Expression of Wnt Signaling Inhibitors/Modulators Increased after UUO

The sFRP and dkk gene families code for proteins that can modulate Wnt signaling and may be part of the normal negative feedback signaling. Dkk1, a negative regulator of Wnt signaling, was identified recently as a direct transcriptional target of β-catenin signaling (20). We determined that dkk1 to 4 and sFRP1 to 5 are expressed in normal kidneys and in the obstructed kidneys (Figure 2, A and B), except for dkk4 and sFRP5, which were not detected in either normal or injured kidneys but were detected in mouse embryos (data not shown). The expression of inhibitor of β-catenin and TCF-4 (ICAT) and β-actin remained constant at all of the UUO time points. It is interesting that the expression of dkk1 and sFRP4 increased between 0 and 4 d of UUO. The increases in dkk1 and sFRP4 were verified by analysis of six normal kidneys and six kidneys that were obstructed for 14 d (Figure 2B). Dkk1 and sFRP4 expression consistently increased after UUO (Figure 2C).

Increased sFRP4 mRNA Expression Occurs in Perivascular Regions

In the normal adult mouse kidney, sFRP4 was weakly expressed in perivascular regions (Figure 3, A, C, and E). After 1 wk of obstruction, the intensity and the area of sFRP4 expression increased (Figure 3, B, D, and F). The sFRP4 was expressed by cells in the adventitial layer (Figure 3E, arrowheads) of the normal renal vasculature, and the number of these sFRP4 expressing cells increased (Figure 3F, arrowheads) after UUO. The sFRP4 expression increased around the vasculature throughout the kidney after UUO.

Total Renal sFRP4 Protein Level Is Decreased after UUO

Despite increased sFRP4 expression, we observed a small decrease in sFRP4 protein levels after UUO (Figure 4, upper sFRP4 panel). With longer exposure, two sFRP4 bands between 41 and 51 kD are visible (Figure 4A, lower sFRP4 panel). The reason for detecting two endogenous forms of sFRP4 is unknown. In normal mouse kidneys, the most prominent location of sFRP4 is in association with peritubular vasculature structures in the renal papillary (Figure 4B) and inner medullary (Figure 4E) regions. This distribution of sFRP4 protein is consistent with the perivascular expression of sFRP4 mRNA. After UUO, there is an increased amount of sFRP4 protein associated with the vasculature (Figure 4, C and F) and is easier to visualize in the bright field (Figure 4D) as a dark brown stain. The increased amount of perivascular sFRP4 protein after UUO is consistent with increased expression of perivascular sFRP4 mRNA.

Apart from the vasculature, sFRP4 protein is localized in cortical tubular structures in normal mouse kidneys (Figure 4G). The cortical tubular sFRP4 protein is unlikely to be of renal origin because the cortical tubular sFRP4 protein levels decrease dramatically after UUO (Figure 4H), whereas renal expression of sFRP4 mRNA increases. The decrease in total sFRP4 protein levels after UUO detected by Western blot analysis is likely due to the decrease in cortical tubular sFRP4 protein levels. UUO triggers tubular epithelial cell de-differentiation, and one manifestation of this may be the disruption of the ability of cortical tubular epithelia to normally sequester circulating sFRP4 protein.

Recombinant sFRP4 Alters the Progression of Fibrosis

We next tested whether administration of recombinant sFRP4 suppresses the injury-induced Wnt-dependent β-catenin signaling and alters the progression of fibrosis. The increase in total fibronectin protein after UUO is modestly suppressed by recombinant sFRP4 (Figure 5, A and B). Mice that received 720 ng or 7.2 μg of recombinant sFRP4 showed a reduction in fibronectin protein levels. The average reduction in fibronectin protein accumulation in the 7-d obstructed kidneys of mice that received 7.2 μg of sFRP4 compared with the vehicle-treated mice was 30% (n = 6; P = 0.0023 by t test; Figure 5C). This reduction in total fibronectin can be attributed to a reduction in the cellular fibronectin (Figure 5A), which is the form deposited in the interstitium during tissue fibrosis and may be critical for the maintenance of differentiated myofibroblasts (21). In turn, the fibrotic matrix production is attributed mainly to myofibroblasts (22,23).

The α-SMA–positive fibroblasts, termed myofibroblasts, increase in number and are partly responsible for the increased synthesis of extracellular matrix during the progression of renal tubulointerstitial fibrosis (2). The α-SMA expression after UUO was variably reduced with the lower dose of recombinant sFRP4 (720 ng) but was substantially and consistently reduced with the higher dose of recombinant sFRP4 (7.2 μg; Figure 5, A and B). On average, the obstructed kidneys of mice that received 7.2 μg of sFRP4 had a 55% reduction in α-SMA expression when compared with vehicle-treated mice (Figure 5C).

In the normal adult mouse kidney, the α-SMA expression is restricted to the vascular smooth muscle cells (VSMC; Figure 6, A and D, arrowheads). In the 7-d obstructed kidneys with vehicle treatment, the α-SMA continues to be expressed by VSMC (Figure 6, B and E, arrowhead) and is also expressed by the increased number of interstitial myofibroblasts (Figure 6, B and E, arrows). The observed increase in α-SMA expression in interstitial cells of vehicle-treated UUO 7-d kidneys is suppressed with administration of 7.2 μg sFRP4 (Figure 6, C and F, arrow), whereas the expression is maintained in the VSMC (Figure 6, C and F, arrowheads). We counted the number of myofibroblasts in four vehicle-treated and four sFRP4-treated 7-d UUO kidneys. There is on average a 60% reduction in the number of myofibroblasts with sFRP4 treatment (Figure 6G). These observations are consistent with the idea that Wnt inhibitors, such as recombinant sFRP4, can suppress the progression of renal fibrosis by reducing the injury-stimulated fibroblast characteristics and activities.

Time Course of Recombinant sFRP4 Accumulation in the Kidney

To determine the time course of recombinant sFRP4 accumulation in obstructed kidneys, we compared obstructed kidneys of vehicle- versus sFRP4-treated mice (Figure 5, A and B). Increased amounts of sFRP4 are present in the obstructed kidneys of mice that were treated with 7.2 μg of recombinant sFRP4 when compared with vehicle-treated mice (Figure 5, A and B). The increased sFRP4 levels are due to increased presence of endogenous and not recombinant sFRP4 (Figure 5D). Two bands are detected between 41 and 51 kD in the obstructed kidney lysates of mice that received recombinant sFRP4 (Figure 5D, lane 1), which migrate slower than the recombinant sFRP4 protein (Figure 5D, lane 2). The mobility differences between the endogenous versus recombinant sFRP4 are likely due to differential posttranslational protein modification, because the recombinant sFRP4 was produced in insect cells (16). The ability of recombinant sFRP4 to maintain endogenous sFRP4 protein levels is further supported by the observed maintenance of cortical tubular sFRP4 in the obstructed kidneys of mice that received recombinant sFRP4 (Figure 4I).

To further support the idea that recombinant sFRP4 is not present in the 7-d obstructed kidneys of mice that had received their final injection of 7.2 μg of recombinant sFRP4 24 h before they were killed, we compared lysates from obstructed kidneys of vehicle-treated mice (Figure 5E, lanes 1 and 2) with that of recombinant sFRP4-treated mice (Figure 5E, lanes 3 through 6). An anti-V5 antibody was used to detect the V5 epitope tag present in recombinant sFRP4 (16). A nonspecific band was detected at approximately 51 kD. Importantly, no bands corresponding to recombinant sFRP4 were detected in the obstructed kidneys 24 h after injection. However, 20 and 40 min after injection of recombinant sFRP4, faint bands corresponding to the size of recombinant sFRP4 were detected (Figure 5E, at the level of the arrow in the last two lanes).

Recombinant sFRP4 Reduces Wnt-Dependent β-Catenin Signaling in Tubular Epithelia after UUO

There is an increase in β-catenin and active β-catenin levels after 7 d of UUO (Figure 1). This increase in β-catenin occurred in epithelial cells of 7-d obstructed kidneys (Figure 7, G through I) and was suppressed with the intraperitoneal administration of recombinant sFRP4 (Figures 5, A and B, and 7, J through L). This finding is consistent with recombinant sFRP4 opposing Wnt-dependent β-catenin signaling in epithelial cells during renal fibrosis. In normal adult mouse kidneys, β-catenin has a basolateral localization within the cortical proximal tubules (Figure 7, A and B) and in other tubular epithelial structures including the inner medullary collecting ducts (Figure 7C). After 4 d of obstruction, there is an increase in cytosolic and nuclear β-catenin in both tubular epithelial (Figure 7, D and E) and interstitial cells (Figure 7F). The increased cytosolic and nuclear β-catenin is evident in 7-d obstructed kidneys of vehicle-treated mice only in tubular epithelial structures (Figure 7, G through I). With recombinant sFRP4 treatment in the 7-d obstructed kidneys, the tubular epithelial β-catenin is restricted primarily to the basolateral membrane, with only a small amount present in the cytoplasm and nucleus. Disruption of β-catenin signaling in the tubular epithelium likely helps to maintain the epithelia in a mature differentiated state. This may be the reason that the cortical tubular epithelia of sFRP4-treated mice retain their ability to sequester endogenous sFRP4 even after 7 d of obstruction.

Injury-Induced Reduction in E-Cadherin mRNA Expression Is Suppressed by sFRP4

We tested whether recombinant sFRP4 protects against injury-induced de-differentiation of tubular epithelia. E-cadherin has frequently been used as a marker of epithelial maturity. In addition, β-catenin/TCF may regulate the repression of E-cadherin expression after UUO, as has been reported in other biologic settings (2426). Western blot analysis using normal and 7-d obstructed kidney lysates revealed ambiguous results. Three bands were detected, including the expected 120-kD E-cadherin band (Figure 8A). The amount of 120-kD E-cadherin did not decrease after UUO. However, an approximately 100-kD band decreased in amount after UUO and was maintained with sFRP4 treatment. The reason for not detecting a decrease in E-cadherin protein levels, like other researchers have reported (27), may be due to our lysis buffer, which is not optimal for isolation of transmembrane proteins. To circumvent these ambiguous results while still using E-cadherin as a marker of epithelial maturity, we measured E-cadherin mRNA levels. We observed by real-time RT-PCR that the relative mRNA levels of E-cadherin are reduced in 4-d obstructed kidneys compared with normal kidneys (Figure 8B). We performed a short-term study with more frequent (once a day starting at the time of obstruction) sFRP4 injections to determine whether recombinant sFRP4 can maintain E-cadherin expression. The reduction in E-cadherin expression is suppressed by sFRP4, as evidenced by the comparison of three vehicle-treated with three sFRP4-treated 4-d obstructed kidneys (Figure 8C).


After Renal Epithelial Injury, There Is an Increase in Wnt-Dependent β-Catenin Signaling Despite Increased Expression of Genes that Code for Wnt Inhibitors

The expression of dkk1 and sFRP4 increased after UUO. The sFRP4 expression increases in perivascular cells and is consistent with the increased sFRP4 protein around the renal vasculature after UUO. However, the overall renal sFRP4 protein levels decreased after UUO. On the basis of our observations, we suggest that circulating sFRP4 is sequestered by normal cortical tubular epithelia and may represent active sFRP4-mediated signaling. After UUO, the overall decrease in renal sFRP4 protein is due to the inability of the injury-modified cortical tubular structures to sequester circulating sFRP4. Despite an increase in renal sFRP4 gene expression, there is an overall decrease in renal sFRP4 protein as a result of the inability of cortical tubules to sequester circulating sFRP4. This in part may contribute to the increased Wnt-dependent β-catenin signaling evident in the cortical tubular epithelia after UUO.

We do not know whether other Wnt inhibitor proteins are similarly decreased after UUO. However, on the basis of the overall renal increase in Wnt-dependent β-catenin signaling, we can conclude that even if Wnt inhibitor proteins are increased after UUO, it is not sufficient to inhibit the increase in Wnt-dependent β-catenin signaling. This may be due to the spatial restriction of the Wnt inhibitor proteins. In addition, the increase in Wnt inhibitors may not be sufficient to compensate for increased Wnt gene expression, such as Wnt4, after UUO.

Recombinant sFRP4 Alters the Progression of Fibrosis by Disrupting Wnt-Dependent β-Catenin Signaling in the Tubular Epithelium

The intraperitoneally injected sFRP4 reaches the cells within the obstructed kidneys by 20 min after injection, begins to decrease in amount by 40 min after injection, and is not detectable 24 h after injection. The recombinant sFRP4 reduces the amount of cytosolic and nuclear β-catenin within the tubular epithelium. This probably occurs by recombinant sFRP4 preventing Wnt proteins from binding with frizzled receptors during UUO. One direct consequence of the sFRP4-mediated suppression of renal epithelial β-catenin levels may be the observed reduction in repression of E-cadherin expression. Protein complexes that contain β-catenin and LEF-1 can bind to a TCF/LEF site within the E-cadherin promoter to repress E-cadherin expression (24,28). In theory, the recombinant sFRP4 could disrupt the interaction between β-catenin and a member of the TCF family to prevent the repression of E-cadherin. Suppression of fibronectin accumulation and myofibroblast number may be a consequence of maintaining the tubular epithelium intact and preventing epithelial to mesenchymal transition (EMT). Alternatively, sFRP4 may directly suppress Wnt-dependent β-catenin signaling within fibroblasts. We observed increased β-catenin staining in interstitial cells of 4-d but not 7-d obstructed kidneys. It is possible that the injections that are given on day 4 after obstruction suppress interstitial β-catenin signaling and lead to the observed reduction in fibronectin and α-SMA in the 7-d obstructed kidneys. Apart from recombinant sFRP4, other inhibitors of Wnt–β-catenin signaling are likely to alter the expression of fibronectin, α-SMA, and hence the progression of fibrosis. In support of this, recombinant DKK1 can decrease α-SMA expression and fibronectin deposition during mouse lung development (29).

Possible Mechanisms by which Wnt-Dependent β-Catenin Signaling Regulates EMT during Renal Fibrosis

The snail/slug and zeb-1/zeb-2 families of zinc finger proteins bind to the E-box DNA elements in the proximal E-cadherin promoter to repress E-cadherin expression (3032). These EMT transcription factors also downregulate other epithelial markers, such as occludin (33), and upregulate the mesenchymal markers fibronectin and vimentin (30). The β-catenin–and TCF/LEF-containing protein complexes may directly activate the expression of these EMT transcription factors. In support of this, the mouse slug promoter can be transactivated by β-catenin (25), and the Xenopus slug promoters contain TCF/LEF binding sites (34). Recently, snail gene expression was shown to be induced by UUO within the renal tubular epithelia in a TGF-β1/Smad3-dependent manner (35). The slug gene is likely to be induced in the renal tubular epithelia after UUO because TGF-β1 induces the expression of slug in cultured primary mouse renal tubular epithelial cells in a Smad3-dependent manner (36). It is likely that increased β-catenin signaling synergizes with the increased TGF-β1/Smad3 signaling after UUO to induce the expression of snail and slug within the renal tubular epithelia. Recombinant sFRP4 and possibly other factors that can reduce β-catenin signaling after renal epithelial injury may potentially suppress the expression of these EMT transcription factors to prevent epithelial de-differentiation and transition into myofibroblasts. Recently, two reports illustrated that Wnt signaling increases the half-life of snail protein, in a manner similar to the regulation of cytosolic β-catenin (37,38). The increased stability of snail protein mediated by Wnt signaling will allow for the repression of E-cadherin expression and triggering of EMT. After renal epithelial injury, Wnt signaling inhibitors may potentially decrease the half-life of snail protein to alter the progression of fibrosis.

Figure 1:
After unilateral ureteral obstruction (UUO), Wnt-dependent β-catenin signaling activity increased, along with markers of fibrosis. Western blot analysis was performed using lysates of normal kidneys from three different mice (lanes 1 through 3) and using obstructed kidney lysates from three 1-wk UUO mice (lanes 4 through 6). The identity of the protein recognized by the primary antibody is indicated on the left-hand side; the protein molecular weight markers are displayed on the right-hand side.
Figure 2:
The relative mRNA levels of Dkk1 and sFRP4 are increased in the UUO kidneys compared with normal kidneys of mice. (A) Reverse transcription–PCR (RT-PCR) was performed on RNA pooled from three different kidneys per lane. In total, six kidneys were analyzed per time point. Thirty cycles of PCR were performed to detect the relative level of expression of each gene. (B) RT-PCR was performed on RNA extracted from six different normal mouse kidneys or from six different obstructed kidneys of 2-wk UUO mice or using tRNA as a negative control. The number of PCR cycles for Dkk1 and sFRP4 were increased to 35, whereas the number of PCR cycles for β-actin was reduced to 25. (C) The relative levels of expression of Dkk1 and sFRP4 mRNA shown in B were quantified using ImageQuant software. The Dkk1 and sFRP4 signals were normalized to that of β-actin, and the average normalized value among the six normal kidneys was set as one relative unit of gene expression. The t test was performed to compare the normal levels of gene expression with that of the 2-wk obstructed kidneys. Both dkk1 and sFRP4 are expressed at significantly higher levels in the 2-wk obstructed kidneys when compared with that of normal mouse kidneys. *P < 0.05.
Figure 3:
The UUO-induced increase in the relative levels of sFRP4 mRNA occurs in the increased number of adventitial cells found in the renal perivascular regions. RNA in situ hybridization was performed using radiolabeled antisense sFRP4 RNA. (A through D) Dark field images in which the yellow grains indicate the location of sFRP4 gene expression. (E and F) Bright field images in which the location of sFRP4 gene expression is visualized as black grains. (A, C, and E) Images of a section through a normal adult mouse kidney. (B, D, and F) Images of a section through a 1-wk obstructed kidney. The lumen of the vascular structure is marked with an “L”; the arrows point at vasculature structures, and the arrowheads point at adventitial cells expressing sFRP4.
Figure 4:
The sFRP4 protein levels are decreased after UUO. (A) Western blot analysis was performed using lysates of normal kidneys from three different mice (lanes 1 through 3) and using obstructed kidney lysates from three 1-wk UUO mice (lanes 4 through 6). The identity of the protein recognized by the primary antibody is indicated on the left-hand side; the protein molecular weight markers are displayed on the right-hand side. In the middle row, the arrow and arrowhead point at the two different forms of sFRP4. (B through I) Immunohistochemistry was performed to determine renal sFRP4 protein distribution in the papillary (B through D), in the inner medullary (E and F), and in the cortical regions (G through I) of kidneys from normal (B, E, and G), UUO 7 d with vehicle-treated (C, D, F, and H), and UUO 7 d with recombinant sFRP4-treated (I) mice. The red fluorescence represents the sFRP4 protein in B through I, except for D; in D, the brown stain represents the sFRP4 protein.
Figure 5:
Recombinant sFRP4 suppresses β-catenin signaling and the induction of markers of fibrosis. (A) Western blot analysis was performed using lysates from the obstructed kidney of two 1-wk UUO mice that were treated with: 0 ng of sFRP4 (lanes 1 and 2), 720 ng of sFRP4 (lanes 3 and 4), and 7.2 μg of sFRP4 (lanes 5 and 6). The identity of the protein recognized by the primary antibody is indicated on the left-hand side; the protein molecular weight markers are displayed on the right-hand side. (B) Western blot analysis was performed using lysates from the obstructed kidneys of more mice: Two 1-wk UUO mice that were treated with 0 ng of sFRP4 (lanes 1 and 2) and two 1-wk UUO mice that were treated with 7.2 μg of sFRP4 (lanes 3 and 4). *The level at which a band of unknown identity appears in lanes 1 and 2, when blotting with anti–active-β-catenin. (C) The 7.2 μg of recombinant sFRP4 administered every 48 h during the 7 d of UUO (n = 6) significantly reduces the injury-induced increase in β-catenin (*P < 0.0008, t test), fibronectin (**P = 0.00023, t test), and α-smooth muscle actin (α-SMA; ***P < 0.0005, t test) when compared with 7-d obstructed kidneys of vehicle-treated mice (n = 6). (D) Western blot analysis to distinguish between recombinant and endogenous sFRP4. Lane 1 contains kidney lysate from a 1-wk obstructed kidney of a mouse that was treated with 7.2 μg of sFRP4, and lane 2 contains 12.7 μg of recombinant sFRP4. (E) Western blot analysis using the anti-V5 antibody to detect the accumulation of recombinant sFRP4 in the obstructed kidneys. The arrow points at two bands present in each of the last two lanes at the expected size of recombinant sFRP4.
Figure 6:
Recombinant sFRP4 suppresses the renal injury–induced increase in the number of α-SMA–positive interstitial cells. (A and D) The α-SMA is detected only in the vascular smooth muscle cells (VSMC) of the normal mouse kidneys. (B and E) After 7 d of UUO in the obstructed kidneys of vehicle-treated mice (n = 4), there is a dramatic increase in the number of interstitial cells that express α-SMA, as pointed out by the arrows. The arrowheads point at α-SMA–positive vascular structures. (C and F) In the obstructed kidneys of 1-wk UUO mice (n = 4) that were treated with 7.2 μg of sFRP4, the number of interstitial cells that were positive for α-SMA staining is reduced (arrow), whereas the α-SMA expression is maintained in the VSMC (arrowheads). (G) The number of myofibroblasts in the 7-d obstructed kidneys of four vehicle-treated versus four sFRP4-treated mice. The average number of myofibroblasts in the obstructed kidneys of the vehicle-treated mice was set at 100 U. *P < 0.05 as determined by t test.
Figure 7:
The increased cytosolic and nuclear β-catenin in the tubular epithelial cells of 7-d obstructed kidneys is suppressed by recombinant sFRP4. The red fluorescence represents β-catenin staining in the cortex (A, B, D, E, G, H, J, and K), and in the inner medullary regions (C, F, I, and L) of kidneys from normal (A through C), UUO 4 d (D through F), UUO 7 d with vehicle-treated (G through I), and UUO 7 d with sFRP4-treated (J through L) mice. The arrowheads point at tubular epithelial cells with increased cytosolic or nuclear β-catenin staining. The arrows point at interstitial cells with increased β-catenin staining.
Figure 8:
The UUO-induced reduction in E-cadherin gene expression is suppressed by sFRP4. (A) Western blot analysis was performed to detect E-cadherin protein expression. The arrow points at the expected 120-kD E-cadherin protein. (B) The average normalized level of E-cadherin expression in the normal mouse kidneys (n=3) was set to 1 U of expression. The height of each bar represents the average relative expression of E-cadherin, which is reduced to approximately 0.6 U in the 4-d obstructed kidneys (n=3). The error bars represent 1 SD. The reduction in E-cadherin expression in the 4-d obstructed kidneys is statistically significant as determined by t test (*P < 0.005). (C) Comparison of the relative amount of E-cadherin expression in the obstructed kidneys of 4-d UUO mice that were treated with vehicle versus recombinant sFRP4 reveals a statistically significant suppression of E-cadherin reduction in the mice that received recombinant sFRP4 treatment as determined by t test (*P < 0.02). When the average relative amount of E-cadherin expression after normalization in the vehicle-treated UUO 4-d kidneys is set at approximately 0.6 U of expression, the average amount of normalized E-cadherin expression in the sFRP4-treated UUO 4-d kidneys is approximately 0.85 U.
Table 1:
Nucleotide sequences of the primers used for reverse transcription–PCR

This work was presented, in part, at the meeting of American Society of Nephrology, St. Louis, Missouri, October 29, 2004.

We thank Crystal Idleburg for assisting with the histology, Frank Strebeck for assisting with the mice, and Helen Odle for assisting in preparation of this manuscript. We thank Dr. Theodore Simon for insightful discussions and for sharing reagents. We also thank Dr. Anna Zuk for helpful suggestions and critically reading this manuscript.

Published online ahead of print.Publication date available at www.jasn.org.


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