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.

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.