Increased Expression of Secreted Frizzled-Related Protein 4 in Polycystic Kidneys

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary disease associated with progressive renal failure. Although cyst growth and compression of surrounding tissue may account for some loss of renal tissue, the other factors contributing to the progressive renal failure in patients with ADPKD are incompletely understood. Here, we report that secreted frizzled-related protein 4 (sFRP4) is upregulated in human ADPKD and in four different animal models of PKD, suggesting that sFRP4 expression is triggered by a common mechanism that underlies cyst formation. Cyst fluid from ADPKD kidneys activated the sFRP4 promoter and induced production of sFRP4 protein in renal tubular epithelial cell lines. Antagonism of the vasopressin 2 receptor blocked both promoter activity and tubular sFRP4 expression. In addition, sFRP4 selectively influenced members of the canonical Wnt signaling cascade and promoted cystogenesis of the zebrafish pronephros. sFRP4 was detected in the urine of both patients and animals with PKD, suggesting that sFRP4 may be a potential biomarker for monitoring the progression of ADPKD. Taken together, these observations suggest a potential role for SFRP4 in the pathogenesis of ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) occurs in approximately one of 1000 humans and causes ESRD in >50% of all affected patients.¹ Cyst formation starts during embryogenesis but typically does not compromise renal function until later in life. Mutations of either PKD1 or PKD2 cause the disease, but why cysts, present in <1% of all nephrons, cause renal failure remains elusive. Persistent proliferation, secretion, and cyst expansion seem to damage the surrounding tissue by reactive changes of the extracellular matrix.² In addition, increased apoptosis of healthy parenchyma seems to contribute to progressive renal failure in human disease.³ Epithelial cells isolated from cystic kidneys show increased levels of proto-oncogene expression, a mislocalization of integral membrane proteins, and active fluid secretion,⁴ but it has remained unclear how these alterations explain the accelerated tissue loss in vivo.

To understand better molecular mechanisms associated with the progression of renal failure in ADPKD, we generated gene profiles of ADPKD kidneys and identified secreted Frizzled-related protein 4 (sFRP4), a member of an evolving family of secreted molecules that antagonize the Wnt signaling cascade,^5,6 as a differentially regulated gene. Members of the sFRP family can directly sequester Wnt,⁷ secreted glycoproteins that bind and activate Frizzled receptors to stabilize β-catenin, and initiate T cell factor/lymphocyte enhancer factor (TCF/LEF)-dependent gene transcription.⁸ The sFRP share a cysteine-rich domain, which mediates homodimerization and interaction with Frizzled receptors to block Wnt binding.⁹ Since abnormal Wnt signaling has been implicated in the pathogenesis of polycystic kidney disease (reviewed in⁸), and a dysregulation of β-catenin degradation results in rapid-onset of polycystic kidney disease,^10,11 we decided to analyze the regulation of sFRP4 expression, the effects of sFRP4 on various components of the Wnt signaling cascade and furthermore examined the effects of sFRP4 overexpression on pronephric kidney development in vivo.

RESULTS

Increased Expression of sFRP4 in Cystic Kidneys of Patients with ADPKD and Animal Models of PKD

Microarray analysis of ADPKD kidneys identified sFRP4 as a differentially regulated gene¹²; reverse transcription–PCR (RT-PCR) confirmed that most ADPKD kidneys express increased amounts of this gene (Figure 1, A and B). We used Pkd2-deficient mice to address the question of whether sFRP4 is upregulated in polycystic kidney disease. Pkd2 (−/−) animals develop renal cysts but die between embryonic day 15 (E15) and E20 of embryogenesis.¹³ Western blot analysis of kidneys from Pkd2 (−/−) mice at E16 revealed increased sFRP4 expression (Figure 1C). To determine whether sFRP4 upregulation was also detectable in animal models of nephronophthisis, an autosomal recessive form of PKD, we also performed Western blot analysis in Invs (−/−) mice. These mice lack functional NPHP2 (Inversin); the corresponding human gene is mutated in the infantile form of nephronophthisis (type II). As shown in Figure 1D, Invs (−/−) mice revealed a similar upregulation of sFRP4, suggesting that excessive sFRP4 production accompanies cyst formation independent of the underlying gene mutation. Cysts typically lose their connection to the draining tubules. Nevertheless, sFRP4 was detectable in the urine of several patients with ADPKD but not in the urine of healthy volunteers (Figure 1E). To investigate urinary sFRP4 further, we examined the excretion of sFRP4 in Han:SPRD rats, a slowly progressive PKD model. The urine of Han:SPRD rats was collected from two different animals at three different time points during a 3-wk interval; the urine was normalized for creatinine and urea concentrations. As shown in Figure 1F, the excretion of sFRP4 increased during the depicted time interval. Because the urine of mice is notoriously difficult to standardize, we determined sFRP4 expression in kidney lysates of pcy mice, a mouse model of type III (NPHP3) nephronophthisis.¹⁴Figure 1G confirmed that sFRP4 concentration increased over time in this animal model of cystic kidney disease as well. Thus, sFRP4 expression is elevated in ADPKD and four different animal models of PKD suggesting that a common final pathway of renal cystogenesis and/or cyst progression induces sFRP4 expression.

Cyst Fluid Stimulates sFRP4 Expression

Cyst fluid contains several growth factors and hormones (e.g., EGF, vasopressin) as well as a lipophilic substance that stimulates the generation of cAMP.^15,16 Because the sFRP4 promoter contains multiple cis-acting elements, including a LEF1-, STAT3-, and cAMP-responsive element binding site,^17,18 we analyzed whether cyst fluid activates the sFRP4 promoter. As shown in Figure 2A, cyst fluid significantly augmented the activity of the sFRP4 promoter fragment −1417/+83. To test whether the cyst fluid–mediated promoter activation is associated with the production of sFRP4 protein, we exposed IMCD cells (a cell line derived from the inner medulla cortical collecting duct) and PT1 cells (a cell line derived from the proximal tubule) to cyst fluid. As shown in Figure 2, B and C, cyst fluid triggered the expression of sFRP4 protein after approximately 2 h of exposure. Both IMCD and PT1 cells responded to cyst fluid within the first 5 d after seeding (Figure 2, B and C, left) but became unresponsive by day 10 (Figure 2, B and C, right). Instead, the 10-d-old cells expressed low basal levels of sFRP4, suggesting that senescent and/or highly differentiated cells become increasingly unresponsive to the stimulating effect of cyst fluid.

Vasopressin 2 Receptor Antagonist SR121463 Inhibits sFRP4 Expression

Vasopressin 2 receptor (V2R) antagonists (V2RA) have been shown to ameliorate the progression of renal disease in mouse models of PKD by blocking the production of cAMP in response to vasopressin.^19,20 Vasopressin (antidiuretic hormone) triggered the expression of sFRP4 in IMCD cells (Figure 3, A and B), albeit only at high concentrations (10 μM). Although IMCD cells are derived from the collecting duct, V2R expression was barely detectable by RT-PCR in the IMCD clone used in this experiments (data not shown). Thus, the low V2R expression may explain why high concentrations of antidiuretic hormone were needed to elicit sFRP4 expression in IMCD cells. Because several hormones and growth factors have been detected in cyst fluid, we examined whether the V2R antagonist SR121463 can block sFRP4 expression triggered by cyst fluid. SR121463, at concentrations as low as 1 nM, almost completely suppressed the cyst fluid–induced sFRP4 expression in IMCD cells (Figure 3C). V2RA ameliorate PKD progression in animal models and are now used in clinical trials to investigate their therapeutic benefit. Because our results indicate that sFRP4 expression can be blocked by V2RA in vitro, we monitored the expression of sFRP4 in response to systemic application of the V2R antagonist in vivo. SR121463 was fed during a 15-wk period to pcy mice. As demonstrated in Figure 3D, V2RA-treated pcy mice expressed less sFRP4 than vehicle-treated pcy mice. Thus, sFRP4 expression seems to correlate with the progression of PKD and responds to V2RA aimed to ameliorate this disease.

sFRP4 Blocks Select Wnt Pathway Components

To understand the consequences of increased sFRP4 expression in PKD, we decided to address the effects of sFRP4 on Wnt signaling. Previous findings demonstrated that sFRP4 abrogates Xenopus Wnt8 (XWnt8)-mediated double-axis formation,²¹ an established system to monitor canonical Wnt activation during Xenopus embryogenesis. We expressed sFRP4 in HEK 293T cells and confirmed that sFRP4 blocks the XWnt8-mediated stabilization of β-catenin, a hallmark of the activated canonical Wnt branch (Figure 4A). sFRP are thought to block canonical Wnt signaling through direct interaction with soluble Wnt molecules.⁷ Consistent with this hypothesis, sFRP4 blocked XWnt8 but had no effect on the stabilization of β-catenin mediated by Dishevelled (Dsh), a cytoplasmic adaptor molecule that acts downstream of Wnt and Frizzled receptors. Interestingly, sFRP4 also had no effect on the stabilization of β-catenin mediated by XWnt3a, suggesting that sFRP4 selectively interferes with some but not all Wnt molecules (Figure 4A). When we analyzed the TCF/LEF-dependent activation of the TOPFLASH luciferase reporter, we confirmed that sFRP4-conditioned medium (sFRP4 CM) blocked XWnt8 but had no effect on XWnt3-mediated activation of the reporter (Figure 4B).

To corroborate our observation further, we monitored the formation of a secondary body axis after ectopic stimulation of the canonical Wnt signaling pathway during early Xenopus embryogenesis. Both XWnt3a and XWnt8 induced a secondary body axis (Figure 5); however, whereas sFRP4 almost completely rescued the phenotypic changes caused by XWnt8, it had no effect on either Dsh- or XWnt3a-mediated axis duplication. This analysis confirms that sFRP4 selectively blocks distinct Wnt molecules. To investigate further mechanisms that mediate the effects of sFRP4 on the canonical Wnt pathway, we analyzed the influence of sFRP4 on rat Frizzled 1 (RFz1)- and Xenopus Frizzled 8 (XFz8)-mediated activation of the canonical Wnt signaling cascade. The stabilization of β-catenin by either RFz1 or XFz8 was blocked by sFRP4 CM, suggesting that sFRP4 inhibits canonical Wnt signaling by binding to both soluble Wnt molecules and Frizzled receptors (Figure 6A). Consistent with this hypothesis, sFRP4 CM blocked the activation of the TOPFLASH reporter, induced by either RFz1 or XFz8 (Figure 6B).

sFRP4 Promotes Cystogenesis of the Zebrafish Pronephros

The zebrafish pronephros is a well-established model system to monitor renal development in vivo.²² To understand the consequences of ectopic sFRP4 expression, we analyzed the phenotypic changes caused by human sFRP4 mRNA during zebrafish embryogenesis. Microinjection of sFRP4 was well tolerated with little mortality (Figure 7A). The Wt1b:GFP transgenic zebrafish line was used to visualize changes of the glomerulus and proximal tubuli by fluorescence microscopy.²³ No pronephric cysts were detectable in control embryos or embryos injected with 50 pg of sFRP4; however, higher amounts (100 and 200 pg) caused clearly detectable pronephric cysts in 6 to 8% of embryos (Figure 7, B and C). Surprising, ectopic expression of sFRP4 also interfered with the development of a normal left–right asymmetry. Heterotaxia of the zebrafish pancreas, observed in <10% of control animals, increased to approximately 30% in zebrafish expressing sFRP4 (Figure 8A). A dorsal body curvature is the hallmark of ciliary defects and characteristic for TRPP2-deficient zebrafish.^24,25 Whereas none of the control animals displayed this abnormality, 30 to 60% of zebrafish embryos injected with ≥50 pg sFRP4 showed an abnormal body curvature (Figure 8B). Taken together, our results suggest that sFRP4 can promote cystogenesis in the zebrafish embryo.

DISCUSSION

Several sFRP molecules are expressed during renal development in the metanephric kidney. In embryonic kidney explants, sFRP1 binds to Wnt4, an essential component of mesenchymal-to-epithelial conversion in the developing kidney, and blocks kidney tubule formation and bud branching.²⁶ In addition to their role in canonical Wnt signaling, sFRP members modulate bone morphogenetic protein signaling during early Xenopus laevis embryogenesis and mediate a cross-talk between hedgehog and Wnt signaling pathways.^27,28 Increased apoptosis rates observed in tissues that express sFRP family members suggest that these molecules promote apoptosis.^29–37 Although the role of apoptosis in ADPKD remains controversial,³⁸ sFRP4 could not only affect the cells lining the cysts but also diffuse to the surrounding tissue and perhaps contribute to the increased apoptotic rates that have been observed in ADPKD kidneys.³ Furthermore, our data indicate that sFRP4 inhibits select members of the canonical Wnt signaling pathway. This selectivity might explain why β-catenin can still accumulate during PKD progression even in the presence of elevated sFRP4 concentrations. Because the sFRP4 promoter region contains a TCF/LEF-binding site and can be stimulated by constitutively active β-catenin,³⁹ it is conceivable that canonical Wnt, not blocked by sFRP4, stimulate sFRP4 production.

Hypermethylation and epigenetic inactivation of sFRP promoter regions has been observed in breast, gastric, renal, and other cancers,^40,41 suggesting that sFRP molecules act as tumor suppressors to curtail Wnt signaling. Their increased expression in some cancers indicates that they are expressed to constrict aberrant Wnt signaling.^42,43 Ciliary defects result in PKD (reviewed in reference⁴⁴) and are associated with a deregulation of Wnt signaling.^45,46 It is conceivable that sFRP4 expression in PKD is caused by an abnormal activation of the Wnt cascade to balance the detrimental effects of uncontrolled Wnt signaling. Alternatively, the damaging effects of progressively expanding cysts may trigger sFRP4 expression. For example, sFRP1 is upregulated after myocardial infarction to limit the consequences of ischemic damage.⁴⁷ Thus, increased sFRP4 expression could represent an adaptive mechanism to protect the kidney from the detrimental effects of progressively expanding cysts; however, the developmental changes caused by sFRP4 expression in zebrafish embryos argue that sFRP4 might represent a disease-promoting factor. Although sFRP4 microinjection is well tolerated, it is associated with pronephric cyst formation, heterotaxia, and an abnormal body curvature. These phenotypic changes are characteristic of zebrafish embryos with ciliary defects²² and suggest that sFRP4 can interfere with cellular programs that maintain normal tubular geometry. The defective left-right patterning in sFRP4-expressing zebrafish may result from an early interference with noncanonical Ca²⁺-dependent Wnt signaling at the dorsal forerunner cells, the precursors that establish the ciliated epithelial cell layer lining the zebrafish Kuppfer's vesicle.⁴⁸ The upregulation of sFRP4 in several animal models of PKD as well as human ADPKD suggests that increased sFRP4 expression is not linked to a particular gene defect. Although sFRP4 is therefore not useful to diagnose a specific form of PKD, monitoring urine and/or blood levels may be helpful to assess the progression of disease in patients with PKD; however, sFRP4 is also detectable in the urine of healthy individuals by mass spectrometry,⁴⁹ and its value as a marker to monitor cystic kidney disease progression needs to be evaluated in large patient collectives with PKD as well as other renal diseases.

CONCISE METHODS

Materials

Patient material was obtained after informed consent. Tissue samples were removed and immediately either snap-frozen in liquid nitrogen for RNA preparation or fixed in 4% PFA. Cyst fluid was collected by aspiration and stored at either 4°C (short-term) or −20°C (long-term). Urine samples were centrifuged at 14,000 rpm for 5 min. The supernatant was stored at either 4°C (short-term) or −20°C (long-term) for further analysis. For RNA isolation, frozen tissue was homogenized in 4 M guanidinium isothiocyanate/0.72% β-mercaptoethanol and subsequently purified on a caesium chloride gradient.

Gene Expression Profiling and Semiquantitative RT-PCR

Microarray analysis and validation by RT-PCR were performed as described.⁵¹ For semiquantitative RT-PCR, total RNA (1 μg) was purified with the RNase free DNase set (Qiagen, Hilden, Germany) and transcribed into cDNAs using an oligo (dT) primer and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). For each PCR reaction, β-actin or GAPDH expression was used as an internal standard control. The amplification cycle consisted of a hot start at 94°C for 2 min, followed by multiple cycles of denaturation at 94°C at 1 min, annealing at 58°C for 1 min, and elongation at 72°C for 1 min. Cycle number was adjusted to mRNA expression level of the different genes. The PCR products were separated on a 2% agarose gel and analyzed in relation to the corresponding control band.

Cell Culture

Conditionally immortalized proximal tubular epithelial (PT) cells were maintained at 33°C in DMEM-F12 supplemented with 2% calf serum. For differentiation of the PT cells, temperature was raised to 37°C. Immortalized mIMCD-3 cells (referred to as IMCD cells) were grown in DMEM - F12 medium supplemented with 10% calf serum. Human embryonic kidney cells (HEK-293T cells) were grown in DMEM supplemented with 10% calf serum.

Western Blot Analysis

Cells were harvested after extensive washing with cold PBS in PBS and lysed in a buffer containing 20 mM Tris-HCL (pH 7.5), 1% Triton X-100, 25 mM NaF, 12.5 mM Na4P2O7, 0.1 mM EDTA, 50 mM NaCl, 2 mM Na₃VO₄, and protease inhibitors. After centrifugation (15,000 g for 15 min, 4°C) and ultra centrifugation steps of the lysates (45,000 rpm for 30 min, 4°C) equal amounts of protein were separated on SDS gels and further processed with β-catenin (Transduction Laboratories, San Jose, CA), γ-tubulin (Sigma, Hamburg, Germany), and β-actin (Cell Signaling, Danvers, MA) antibodies for Westen blot analysis. The sFRP-4 monoclonal antibody was recently described.⁵⁰

Reporter Assays

Reporter assays were performed using Great EscAPe SEAP chemiluminescence detection kit (BD Bioscience, Heidelberg, Germany). The results are presented as relative luciferase or alkaline phosphatase activities after normalization for β-galactosidase activity.

Genotyping and Organ Preparation

Kidneys were explanted from Pkd2-, Invs- or pcy -mice at E16 (Pkd2), E20 (Invs) or and processed for Western blot analysis immediately after organ removal. Additionally, material from the head was used for genotyping. For Western blot analysis, the kidneys were homogenized and lysed for 30 minutes on ice in a buffer containing 1% Triton and 0.1 mM EDTA. Equal amounts were used for Western blot analysis. Pkd2 and Invs E16 and E20 embryos were genotyped by PCR. The following primers were used to genotype Pkd2-mice: 5′-CCCATGGCGATGCCTGCTTGCCG-3′, 5′-GGCGATAGAAGGCGATGCGCTGCG-3′ (neomycin cassette), 5′-CGTCCAATGAATTTGCACCAACAAGAACGC-3′, 5′-CTTTCGTCCTGCTCCAGGCAAGCGGAGC-3′ (to amplify a Pkd2 specific region to distinguish between wild-type, heterozygote, and homozygote animals) and Invs mice: 5′-GATTACGTAATAGTGGTCCCTCAGG-3′, 5′-CTGTCCAGTGCACCATGTGGACCT-3′ (thymidine kinase cassette), and 5′-GTATTTACTCAGTGGCCTCAG-3′, 5′-CCGATCACAGGATTGCTAG-3′ (to amplify exon 3).

Zebrafish Microinjections

Microinjections and analysis were performed as recently described.²⁵ Briefly: zebrafish embryos were microinjected at the 1 to 2 cell stage with various amounts of synthesized capped human sFRP4 mRNA diluted in a solution containing 200 mM KCL, 0.1% Phenol Red, and 10 mM HEPES (pH 7.5). Embryos were dechorionated manually at 24–30 hours post fertilization (hpf), and were kept at 28.5°C in Danieau's solution with 0.003% 1-Phenyl-2-thiourea (Sigma) to suppress pigmentation. Staging was done according to hpf. Zebrafish embryos were analyzed under a Leica MZ16 stereo-microscope (Leica, Solms, Germany). Pictures were taken with a SPOT Insight Fire Wire System and processed with the SPOT Imaging Software (Diagnostic Instruments, Sterling Heights, MI).

DISCLOSURES

None.

Figure 1: Increased sFRP4 expression in ADPKD kidneys. (A) Microarray analysis of total RNA extracted from ADPKD kidneys revealed that sFRP4 expression normalized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was increased in comparison with tissue from normal kidneys. Two ADPKD kidneys could not be analyzed because of spotting errors. (B) The microarray data were confirmed by semiquantitative RT-PCR. (C and D) Western blot analysis of whole-kidney lysates from Pkd2 (−/−) at E16 (C) and INVS-deficient mice at E19 (D) demonstrated that sFRP4 was upregulated in kidneys of homozygote knockout embryos, compared with heterozygotes or wild-type mice. γ-Tubulin was used as a loading control. PCR primers directed against the neomycin cassette (Neo) or the thymidin kinase cassette (TK) as well as gene-specific primers were used to distinguish from among wild-type, heterozygote, and homozygote mice. (E) The urine of six patients with ADPKD (lanes 1 through 6) but not the urine of healthy individuals (lanes A through C) contained hsFRP4. (F) Urine samples from two different heterozygote, male Han:SPRD rats (Cy/+) were collected at postnatal weeks 6, 9, and 12 (corresponding to lanes 1 through 3). The samples were standardized to contain the same amount of either creatinine or urea, demonstrating that sFRP4 excretion in the urine increases with the progression of the disease. (G) Equal amounts of protein (1 μg) were obtained from pcy mice kidney lysates, ranging from 10 to 56 wk of age. Analysis by Western blot revealed increased sFRP4 protein level over time; actin was used to control for equal loading.

Figure 2: The sFRP4 promoter is activated by cyst fluid in a concentration-dependent manner. (A) HEK 293T cells were transfected with the sFRP4 promoter fragment −1417 to +83, driving the expression of a secreted alkaline phosphatase and β-galactosidase, to normalize for transfection efficiency. Twelve hours after transfection, the cells were incubated with diluted cyst fluid (CF) as indicated; the promoter activity was assayed 4 h later. All experiments were performed in triplicate and repeated at least three times. (B and C) IMCD (B) or PT1 (C) cells were stimulated with CF after 5 d of culture (left) or after 10 d of culture (right) and assayed for sFRP4 expression by Western blot analysis; γ-tubulin was used as a loading control.

Figure 3: Vasopressin and the V2RA SR121463 affect sFRP4 expression levels in vitro and in vivo. (A) Expression of sFRP4 in response to increasing concentrations of vasopressin (VP; 0.1 to 10.0 μM) in IMCD cells; γ-tubulin served as a loading control. (B) Time course of sFRP4 expression in response to 10 μM vasopressin; γ-tubulin served as a loading control. (C) The V2RA SR121463 blocked sFRP4 expression, triggered by CF in IMCD cells. (D) Kidneys of two pcy mice (lanes 1 and 2), treated with SR121463 over a period of 15 wk, and two vehicle treated pcy mice (lanes 3 and 4) were explanted and homogenized. Equal amounts of protein were analyzed on Western blot and compared for sFRP4 concentration. Results showed reduced amounts of sFRP4 expression in treated animals in comparison with vehicle-treated mice. Actin was used as a loading control.

Figure 4: sFRP4 blocks XWnt8- but not XWnt3a-mediated activation of the canonical Wnt pathway. (A) HEK 293T cells were transfected with equal amounts (2 μg) of pcDNA3 vector, XWnt8, Dsh, and XWnt3a and incubated with or without sFRP4-CM for 12 h. Accumulation of cytoplasmic β-catenin was used to monitor the activation of canonical Wnt signaling; γ-tubulin served as a loading control. (B) HEK 293T cells were co-transfected with the TOPFLASH reporter construct and β-galactosidase, to normalize for transfection efficiency, in combination with a control (CTL) plasmid, XWnt8, Dsh, or XWnt3a and incubated with or without sFRP4-CM for 12 h.

Figure 5: sFRP4 blocks double-axis formation caused by XWnt8 but has no effect on XWnt3a-mediated activation of the canonical Wnt signaling cascade in Xenopus embryogenesis. Xenopus laevis eggs were injected dorsolaterally at the four-cell stage with Dsh, XWnt3a, XWnt8, and sFRP4 mRNA as indicated and scored at tadpole stage 37/38. Whereas sFRP4 blocked the formation of a secondary axis mediated by XWnt8, the XWnt3a-induced secondary axis was not rescued by co-injection of sFRP4 mRNA. The percentages of no axis duplication and partial or complete axis duplication are shown in the bars; numbers of scored tadpoles are given on top of each bar.

Figure 6: sFRP4 inhibits Frizzled-mediated activation of the canonical wnt signaling branch. (A) HEK 293T cells were transfected with pCDNA6 (CTL), RFz1, or XFz8 and incubated with sFRP4-CM for 12 h as indicated. Cytoplasmic β-catenin levels were used to monitor the activation of the canonical Wnt pathway; γ-tubulin served as a loading control. The Frizzled-mediated accumulation of cytosolic β-catenin was suppressed by sFRP4. (B) HEK 293T cells were co-transfected with the TOPFLASH reporter construct and pcDNA6 (CTL), RFz1, or XFz8; β-galactosidase was used to normalize for transfection efficiency. sFRP4-CM inhibited the RFz1- and XFz8-mediated activation of the TOPFLASH reporter construct.

Figure 7: sFRP4 promotes cyst formation in the zebrafish pronephros. (A) Zebrafish embryos were injected with 50, 100, and 200 pg of human sFRP4, and survival was monitored at 5 to 7, 24, and 55 h postfertilization (hpf). Human sFRP4 was well tolerated and not associated with substantial mortality (CTL, uninjected control embryos). Depicted is the number of surviving embryos. (B) Cysts, as indicated by the asterisks, were detectable within the proximal tubuli adjacent to the single zebrafish glomerulus. Arrows point to pronephric ducts. The Wt1b:GFP transgenic zebrafish line was used to visualize the proximal pronephros by fluorescence microscopy (CTL, control embryos). (C) Injection of sFRP4 (100 to 200 pg) resulted in 6 to 8% pronephric cysts (CTL, control embryos).

Figure 8: Ectopic expression of sFRP4 causes heterotaxia and an abnormal body curvature in zebrafish embryos. (A) The Wt1b:GFP transgene labels the pancreas of zebrafish embryo (dorsal view, anterior to the top). Microinjection of sFRP4 increased the frequency of pancreatic heterotaxia (arrow) from ≤10% in control Wt1b:GFP transgenic zebrafish (CTL) to 30% in zebrafish injected with 50 pg of sFRP4; zebrafish were analyzed at 72 hpf. (B) Dorsal body curvature, not observed in control animals, was present in 30 to 60% of animals injected with ≥50 pg of sFRP4. To quantify the changes, microinjected zebrafish embryos were grouped into three dysmorph classes (Dys I through III) according to the degree of body curvature abnormalities.

This study was supported by Deutsche Forschungsgesellschaft grant WA 597/10 to G.W.

We thank the members of the Renal Division for helpful discussion. We gratefully acknowledge the gift of the vasopressin 2 receptor antagonist SR121463, provided by Sanofi-Aventis. Han:SPRD rats were kindly provided by N. Gretz, Mannheim, and the sFRP4 promoter constructs by W.L. Wendy Hsiao, Hong Kong.