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Wnt/␤-Catenin Signaling Promotes Renal Interstitial Fibrosis

Weichun He,*† Chunsun Dai,* Yingjian Li,* Gang Zeng,* Satdarshan P. Monga,* and Youhua Liu*

*Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; †Department of Medicine, The Second Affiliated Hospital, Nanjing Medical University, Nanjing, China

ABSTRACT Wnts compose a family of signaling that play an essential role in kidney development, but their expression in adult kidney is thought to be silenced. Here, we analyzed the expression and regulation of Wnts and their receptors and antagonists in normal and fibrotic kidneys after obstructive injury. In the normal mouse kidney, the vast majority of 19 different Wnts and 10 was expressed at various levels. After unilateral ureteral obstruction, all members of the Wnt family except Wnt5b, Wnt8b, and Wnt9b were upregulated in the fibrotic kidney with distinct dynamics. In addition, the expression of most Fzd receptors and Wnt antagonists was also induced. Obstructive injury led to a dramatic accumulation of ␤-catenin in the cytoplasm and nuclei of renal tubular epithelial cells, indicating activation of the canonical pathway of Wnt signaling. Numerous Wnt/␤-catenin target genes (c-Myc, Twist, lymphoid enhancer-binding factor 1, and fibronectin) were induced, and their expression was closely correlated with renal ␤-catenin abundance. Delivery of the Wnt antagonist Dickkopf-1 significantly reduced renal ␤-catenin accumu- lation and inhibited the expression of Wnt/␤-catenin target genes. Furthermore, gene therapy with Dick- kopf-1 inhibited myofibroblast activation; suppressed expression of fibroblast-specific 1, type I collagen, and fibronectin; and reduced total collagen content in the model of obstructive nephropathy. In summary, these results establish a role for Wnt/␤-catenin signaling in the pathogenesis of renal fibrosis and identify this pathway as a potential therapeutic target.

J Am Soc Nephrol 20: 765–776, 2009. doi: 10.1681/ASN.2008060566

The Wnt family of secreted signaling proteins plays hancer-binding factor (LEF) to stimulate the tran- an essential role in organogenesis, tissue homeosta- scription of Wnt target genes.5–7 In addition to this sis, and tumor formation.1–4 Aberrant regulation of canonical pathway, Wnt proteins may exert their Wnt signaling has been implicated in the pathogen- activities through numerous ␤-catenin–indepen- esis of many human diseases in diverse types of tis- dent, noncanonical intracellular signaling routes.8 sues.3,4 Wnt proteins transmit their signal across the Both Wnts and Fzd receptors are encoded by plasma membrane through interacting with ser- multiple, distinct genes, creating a complex net- pentine receptors, the Frizzled (Fzd) family of pro- work of signaling system with enormous degree of teins, and co-receptors, members of the LDL recep- tor–related protein (LRP5/6). Upon binding to Received June 4, 2008. Accepted October 28, 2008. their receptors, Wnt proteins induce a series of downstream signaling events involving Disheveled Published online ahead of print. Publication date available at www.jasn.org. (Dvl), axin, adenomatosis polyposis coli, and glyco- ␤ Correspondence: Dr. Youhua Liu, Department of Pathology, Uni- gen synthase kinase 3 , resulting in dephosphory- versity of Pittsburgh, S-405 Biomedical Science Tower, 200 Lo- lation of ␤-catenin. This leads to the stabilization of throp Street, Pittsburgh, PA 15261. Phone: 412-648-8253; Fax: ␤-catenin, rendering it to translocate into the nu- 412-648-1916; E-mail: [email protected] clei, where it binds to T cell factor/lymphoid en- Copyright ᮊ 2009 by the American Society of Nephrology

J Am Soc Nephrol 20: 765–776, 2009 ISSN : 1046-6673/2004-765 765 BASIC RESEARCH www.jasn.org diversity as well as redundancy. At least 19 distinct Wnt proteins and 10 different Fzd receptors have been identified in mouse9,10 (see The Wnt Homepage http://www. stanford.edu/ϳrnusse/wntwindow.html). Not surprising, Wnt signaling is tightly reg- ulated in a multitude of ways. There are several secreted antagonists of Wnt signal- ing, including soluble Frizzled-related pro- tein (sFRP), Wnt inhibitory factor, and a family of Dickkopf (DKK) proteins.1,5 Of them, DKK proteins are unique in that they specifically inhibit the canonical Wnt signal pathway by binding to the LRP5/6 compo- nent of the receptor complex.11,12 Wnt/␤-catenin signaling has been shown to play a role in kidney development and dis- eases. Wnt4 and Wnt9b are highly expressed in the early stage during kidney develop- ment and are functionally important for nephron formation.13,14 In adult kidney, however, Wnt signaling seems to be si- lenced.14–16 Dysregulation of Wnt/␤-cate- nin signaling occurs in certain types of kid- ney diseases, including obstructive neph- 17,18 ropathy. These observations clearly sug- Figure 1. Expression of Wnt genes in normal and fibrotic mouse kidneys. (A) Repre- gest a potential role of Wnt signaling in mam- sentative RT-PCR results show the expression of different Wnt genes in normal mouse malian nephrogenesis, tissue homeostasis, kidney. In the absence of RT, no PCR product was detected, suggesting the specificity. and pathogenesis of kidney diseases; how- (B) Representative RT-PCR results demonstrate the steady-state levels of renal Wnt ever, the expression of 19 Wnts and 10 Fzd mRNA at different time points after UUO as indicated. Numbers (1, 2, and 3) indicate receptors in adult kidney remains to be deter- each individual animal in a given group. (C) Graphic presentation shows the distinct, mined. Furthermore, their regulation and dynamic pattern of Wnt regulation in the fibrotic kidney. Different Wnts with similar function in the evolution of chronic kidney dynamic pattern after injury were grouped. The actual values of relative mRNA levels diseases are poorly understood. (fold induction over sham controls) are presented in Supplemental Table 1. In this study, we performed a compre- hensive analysis of the expression and regulation of Wnts and 1A, the vast majority of 19 Wnts, except Wnt3a, Wnt8a, and their receptors and antagonists in normal and fibrotic kidneys Wnt10b, were expressed at different levels in mouse adult kid- after obstructive injury. Our data indicate that the majority of ney. In the absence of RT, no PCR product was detected, sug- Wnts and Fzd receptors are upregulated in diseased kidney, gesting the specificity of Wnt expression. We next investigated which leads to a dramatic accumulation of ␤-catenin, resulting the regulation of Wnt expression during the course of renal in induction of the Wnt/␤-catenin target genes. Furthermore, interstitial fibrosis induced by unilateral ureteral obstruction we show that delivery of Wnt antagonist DKK1 gene reduces (UUO). As shown in Figure 1B, the steady-state mRNA levels ␤-catenin accumulation and attenuates renal interstitial fibro- of most Wnt genes were increased at different time points after sis in a mouse model of obstructive nephropathy. These studies UUO. The actual values of renal mRNA levels of various Wnts establish a critical role of hyperactive Wnt/␤-catenin signaling are presented in the Supplemental Table 1. On the basis of the in the pathogenesis of renal fibrosis and present a novel target characteristic features of Wnt regulation, four dynamic pat- for therapeutic intervention of fibrotic kidney diseases. terns of Wnt expression during the process of renal fibrosis could be classified. As presented in Figure 1C, there were only three Wnts, including Wnt5b, Wnt8b, and Wnt9b, whose ex- RESULTS pression was unaltered throughout the course of renal fibro- genesis after UUO. Wnt1, Wnt7a, and Wnt7b displayed a sim- Expression of Wnt Genes in Normal and Fibrotic Kidneys ilar expression pattern, with a peak induction at 7 d after We first performed a systematic analysis of the mRNA expres- obstructive injury, followed by declining in mRNA levels. The sion of all Wnt genes in normal mouse kidney by the reverse expression of five other Wnts, including Wnt2b, Wnt3, Wnt5a, transcriptase–PCR (RT-PCR) approach. As shown in Figure Wnt9a, and Wnt16, was initially increased up to 7 d and sus-

766 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 765–776, 2009 www.jasn.org BASIC RESEARCH

tained thereafter. The remaining eight members of Wnt family shared a comparable induction dynamic, with a continuous increase in mRNA expression during the entire experimental period. Of interest, there was no single Wnt whose expression was suppressed in the fibrotic kidney after UUO. Wnt protein was also upregulated in the fibrotic kidney. Figure 2 shows the protein levels of Wnt4 and Wnt7a at differ- ent time points after UUO. Similar to their mRNA, substantial increase in renal Wnt4 and Wnt7a protein abundance was ev- ident in a time-dependent manner. Of note, Wnt4 and Wnt7a protein also exhibited distinct patterns of induction dynamics (Figure 2). To address whether Wnt induction is a general phe- nomenon in renal fibrogenesis, we also examined Wnt expres- sion in a mouse model of adriamycin nephropathy. As pre- Figure 2. Induction of renal Wnt4 and Wnt7a protein expres- sented in Supplemental Figure 1, the expression of many Wnts sion after obstructive injury. Whole-kidney homogenates were was also upregulated in diseased kidney at 5 wk after injection prepared at different time points after UUO as indicated and of adriamycin, a time point when significant glomerular and immunoblotted with antibodies against Wnt4, Wnt7a, and interstitial fibrosis is evident.19 glyceraldehyde-3-phosphate dehydrogenase (GAPDH), re- spectively. Numbers (1 and 2) indicate each individual animal Regulation of Wnt Receptors and Antagonists in a given group. We next examined the expression of the Fzd receptor genes in mouse kidney. As shown in Figure 3A, except for Fzd10, the

Figure 3. Regulation of the Fzd receptor genes in normal and fibrotic kidneys. (A) Representative RT-PCR results show the expression of different Fzd receptor genes in mouse kidney. In the absence of RT, no PCR product was detected, suggesting the specificity. (B) Representative RT-PCR results demonstrate the steady-state levels of renal Fzd mRNA at different time points after UUO as indicated. Numbers (1, 2, and 3) indicate each individual animal. (C) Graphic presentation shows the dynamic pattern of Fzd regulation in the fibrotic kidney. Different Fzd receptors with similar expression pattern after injury were grouped. The actual values of relative mRNA levels (fold induction over sham controls) are presented in Supplemental Table 2.

J Am Soc Nephrol 20: 765–776, 2009 Wnt/␤-Catenin and Renal Fibrosis 767 BASIC RESEARCH www.jasn.org mRNA of all Fzd genes (Fzd1 through 9) could be detectable in Activation of Wnt/␤-Catenin Canonical Pathway in mouse adult kidney by the RT-PCR approach, albeit different Obstructive Nephropathy in abundance. Figure 3B shows the representative RT-PCR re- To examine the functional consequence of Wnt regulation in sults of renal Fzd mRNA levels at different time points after renal fibrosis, we next sought to investigate the activation of UUO. The actual values of relative Fzd mRNA levels are pre- ␤-catenin, the principal mediator of the canonical pathway of sented in Supplemental Table 2. As shown in Figure 3C, the Wnt signaling. Figure 5, A and B, demonstrated the expression expression of Fzd4 and Fzd5 was not changed throughout the and localization of ␤-catenin protein in normal and obstructed period of the experiments. Fzd1, Fzd2, Fzd6, Fzd7, Fzd8, and kidney at 7 d after UUO. Compared with the sham controls, Fzd9, however, were moderately induced, whereas the expres- ␤-catenin protein was clearly upregulated predominantly in sion of Fzd3 and Fzd10 genes was significantly upregulated renal tubules of the obstructed kidney, as shown by immuno- (Figure 3C). Once again, none of the Fzd genes was repressed histochemical staining. Besides at the sites of cell–cell adhe- in fibrotic kidney after obstructive injury. sFRP (or Frzb), a sions, ␤-catenin was localized in the cytoplasm and the nuclei Wnt antagonist, was not significantly changed throughout the of tubular epithelial cells (Figure 5B, boxed area). Of note, experiments (Figure 3C). ␤-catenin–positive cells were also observed in the interstitium We further investigated the expression of a family of Wnt (Figure 5B, arrows). Judging from the shape and size of the antagonist DKK genes in normal and fibrotic kidneys. Figure nuclei, it is likely that these interstitial ␤-catenin–positive cells 4A shows the representative RT-PCR results of the steady-state were the estranged tubular cells. Western blot analysis also levels of various DKK mRNA at different time points after revealed a dramatic increase in renal ␤-catenin abundance af- UUO. All four members of the DKK family proteins were ex- ter obstructive injury (Figure 5C). Relative ␤-catenin levels pressed in normal mouse kidney, and their mRNA levels were were increased by approximately four-fold over the sham con- moderately increased after ureteral obstruction (Figure 4A). trols at 14 d after UUO (Figure 5D), suggesting that induction Analysis of the expression dynamics revealed that DKK1 and of Wnt expression would result in an accumulation of ␤-cate- DKK2 expression peaked at 7 d after UUO and declined there- nin in fibrotic kidney. after, whereas the abundances of DKK3 and DKK4 mRNA We further studied the expression of several putative target were induced slightly and gradually throughout the experi- genes of Wnt/␤-catenin signaling in obstructive nephropathy. ments (Figure 4B). As shown in Figure 6A, numerous widely known Wnt/␤-cate- nin target genes, including Twist, LEF1, and fibronectin, were upregulated in the obstructed kidney in a time-dependent manner. The steady-state mRNA levels of these genes were closely correlated with the abundance of renal ␤-catenin throughout the experiments (Figure 6, B through D). In addi- tion, c-Myc and Twist proteins were dramatically increased in the kidney after obstructive injury (Figure 6, E through H).

DKK1 Gene Therapy Blocks Wnt/␤-Catenin Signaling To block Wnt signaling, we used a hydrodynamic-based gene delivery approach20,21 by intravenous injection of na- ked plasmid vector encoding DKK1, a secreted Wnt antag- onist that specifically blocks the canonical pathway of Wnt/ ␤-catenin signaling. As shown in Figure 7, A and B, delivery of DKK1 gene by this approach resulted in substantial ex- pression of exogenous DKK1 transgene in liver and kidney, as revealed by RT-PCR analysis using human-specific prim- ers. Quantitative ELISA that detects both human and en- dogenous mouse DKK1 also showed an increased DKK1 protein in liver and kidney after plasmid injection (Figure 7, C and D). Similarly, human Flag-tagged DKK1 protein was detectable in kidney with specific anti-Flag antibody (Figure E). Because it is a secreted protein, circulating DKK1 level also Figure 4. Expression of Wnt antagonists in obstructive nephrop- elevated markedly after plasmid injection (Figure 7F). athy. (A) Representative RT-PCR results demonstrate the steady- We found that delivery of DKK1 gene significantly inhib- state levels of various Dickkopf (DKK1 through 4) mRNA at dif- ␤ ferent time points after UUO as indicated. Numbers (1, 2, and 3) ited the stabilization and accumulation of -catenin in ob- indicate each individual animal. (B) Graphic presentation shows structed kidneys (Figure 8, A through C). Compared with the dynamic pattern of DKK regulation in the fibrotic kidney. the empty vector controls (Figure 8B), renal ␤-catenin pro- DKKs with similar expression pattern after injury were grouped. tein, as shown by immunohistochemical staining, was re-

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Figure 5. Activation of the Wnt/␤-catenin canonical pathway in obstructive nephropathy. (A and B) Representative micrographs demonstrate the accumulation and localization of ␤-catenin in fibrotic kidney. Kidneys from sham (A) and UUO for 7 d (B) were stained immunohistochemically for ␤-catenin protein. Bar ϭ 40 ␮m. Arrows indicate ␤-catenin–positive cells in the interstitium. Arrowheads in the enlarged box area indicate positive nuclear staining. (C and D) Western blot analysis shows a dramatic increase in renal ␤-catenin abundance after obstructive injury. Representative Western blot (C) and quantitative data (D) are presented. Relative ␤-catenin levels (fold induction over sham controls) were reported after normalizing with GAPDH. Data are means Ϯ SEM of five animals per group. *P Ͻ 0.05, **P Ͻ 0.01 versus sham controls. duced after exogenous DKK1 expression (Figure 8C). Con- We also investigated the expression of fibroblast-specific sistent with a decreased ␤-catenin level, the numbers of cells protein 1 (FSP-1), also known as S100A4 protein, which is with positive cytoplasmic and nuclear staining of ␤-catenin normally expressed in fibroblasts but not epithelia.22 As shown were reduced as well. Western blot analysis also showed that in Figure 9E, very few FSP-1–positive cells were detected in the delivery of DKK1 gene reduced renal ␤-catenin abundance interstitium of normal kidney, as shown by immunohisto- after obstructive injury (Figure 8, D and E). Furthermore, chemical staining. Obstructive injury apparently caused a ectopic expression of DKK1 significantly inhibited the ex- marked induction of FSP-1 expression, and an increased num- pression of several Wnt/␤-catenin target genes such as c- ber of interstitial and tubular epithelial cells became positive for Myc and Twist (Figure 8). Hence, DKK1 gene therapy effec- FSP-1 staining; however, delivery of DKK1 gene not only reduced tively impedes the canonical pathway of Wnt/␤-catenin the overall numbers of renal FSP-1–positive cells (Figure 9F) but signaling in diseased kidney. also particularly inhibited tubular expression of FSP-1 (Figure 9E). Blockade of Wnt Signaling Inhibits Renal ␣-Smooth Muscle Actin and Fibroblast-Specific Protein 1 Blockade of Wnt Signaling Reduces Renal Fibrosis and Expression after Obstructive Injury Inhibits Interstitial Matrix Production We next examined the effects of blockade of Wnt signaling Figure 10 shows that inhibition of Wnt signaling by DKK1 on myofibroblast activation in the evolution of interstitial attenuated renal interstitial fibrosis after UUO. Picrosirius red fibrosis after obstructive injury. As shown in Figure 9, A and and Masson trichrome staining revealed a reduced interstitial B, UUO caused a dramatic induction of the mRNA expres- injury and collagen deposition in the obstructed kidney after sion of ␣-smooth muscle actin (␣-SMA), the molecular sig- ectopic expression of exogenous DKK1 (Figure 10, A through nature of myofibroblasts; however, delivery of DKK1 gene F). This reduction of collagen deposition was associated with a significantly suppressed renal ␣-SMA mRNA expression af- decreased renal interstitial volume in the obstructed kidney ter injury. Ectopic expression of DKK1 also inhibited after exogenous DKK1 expression (Figure 10G). Biochemical ␣-SMA protein expression in obstructed kidney (Figure 9, C analysis of tissue hydroxyproline content demonstrated that and D). Similar results were obtained when the kidney sec- blockade of Wnt signaling by DKK1 attenuated total collagen tions were immunostained with antibody against ␣-SMA in the obstructed kidney, compared with controls (Figure (Figure 9E). 10H).

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Figure 6. Expression of Wnt/␤-catenin target genes in obstructive nephropathy. (A) RT-PCR analysis shows the induction of putative Wnt/␤-catenin target genes in the obstructed kidney at different time points after UUO. (B through D) Linear regression shows a close correlation between renal Twist (B), LEF1 (C), and fibronectin (D) mRNA levels and ␤-catenin abundance (arbitrary units). The correlation coefficients (R2) are shown. (E through H) Western blot analyses demonstrate a dramatic increase in renal c-Myc and Twist protein abundance after obstructive injury. Representative Western blot (E and G) and quantitative data (F and H) are presented. Relative c-Myc and Twist protein levels (fold induction over sham controls) are reported after normalization with GAPDH. Data are means Ϯ SEM of five animals per group. *P Ͻ 0.05, **P Ͻ 0.01 versus sham controls.

We then investigated the expression of type I collagen and the expression of its downstream target genes. Furthermore, fibronectin, two major components of interstitial matrix. As blockade of the canonical pathway of Wnt signaling by DKK1 shown in Figure 11, A through D, obstructive injury induced a inhibits interstitial matrix and attenuates collagen marked increase in the mRNA levels of type I collagen and deposition and tissue scarring. Our studies suggest that the Wnt/ fibronectin, and delivery of DKK1 gene significantly sup- ␤-catenin signaling is hyperactive and detrimental in the evolu- pressed the expression of these matrix components. Similar tion of renal interstitial fibrosis. These findings provide significant results on type I collagen and fibronectin expression were also insights into the role and mechanisms of Wnt/␤-catenin signaling obtained by immunofluorescence staining. It seems clear that in renal fibrogenesis and offer a new strategy in developing ther- blocking the canonical pathway of Wnt/␤-catenin signaling apeutic modalities for the treatment of fibrotic kidney diseases. inhibits interstitial matrix gene expression and attenuates renal Wnt/␤-catenin signaling is generally considered to be fibrotic lesions. silenced in adult tissues.1,5,16 It seems clear, however, that the vast majority of the members of both Wnt and Fzd re- ceptor family genes are expressed at different levels in DISCUSSION mouse adult kidney. This somewhat surprising finding sug- gests that Wnt signaling is important in the maintenance of The results reported here represent the first systematic analysis renal cell and tissue homeostasis under normal physiologic of the expression and regulation of all members of the Wnt conditions. Besides the well-characterized canonical path- family and their Fzd receptor genes in normal and fibrotic way,5,9 in which ␤-catenin is the principal mediator, Wnt kidneys. We demonstrate that Wnt upregulation after chronic signals can be transmitted through several additional intra- renal injury leads to accumulation of ␤-catenin and induces cellular, ␤-catenin–independent, noncanonical pathways,

770 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 765–776, 2009 www.jasn.org BASIC RESEARCH

Figure 7. Expression of exogenous DKK1 after gene therapy. (A and B) RT-PCR analysis demonstrates the expression of exogenous DKK1 gene in liver (A) and kidney (B) after plasmid injection. Total RNA was prepared from liver and kidney at 16 h after DKK1 plasmid injected and subjected to RT-PCR analysis for human DKK1 expression. Numbers (1, 2, and 3) indicate each individual animal in a given group. (C and D) ELISA analysis shows an increased DKK1 protein in liver (C) and kidney (D) after gene delivery. DKK1 protein levels in liver and kidney homogenates were expressed as ng/mg total protein. *P Ͻ 0.05 versus pcDNA3 controls (n ϭ 4 to 5). (E) Western blot analysis demonstrates renal expression of exogenous DKK1 protein. Kidney homogenates were immunoblotted with anti-Flag and anti-GAPDH antibodies, respectively. (F) ELISA analysis shows an increased DKK1 protein in the circulation after gene delivery. Plasma samples were collected from the mice at 16 h after DKK1 plasmid injection, and DKK1 protein levels were expressed as ng/ml. *P Ͻ 0.05 versus pcDNA3 controls (n ϭ 3).

Figure 8. Delivery of DKK1 gene blocks Wnt/␤-catenin signaling in obstructive nephropathy. (A through C) Representative micro- graphs demonstrate renal ␤-catenin expression and localization in sham mice (A), mice that underwent UUO and were administered an injection of pcDNA3 (B), and mice that underwent UUO and were administered an injection of pFlag-DKK1 plasmid (C). Bar ϭ 40 ␮m. (D through F) Representative Western blots (D) and quantitative data (E and F) show that delivery of DKK1 gene reduced renal ␤-catenin and c-Myc abundance after obstructive injury. **P Ͻ 0.01 versus sham controls; †P Ͻ 0.05 versus pcDNA3 (n ϭ 4 to 5). (G through I) DKK1 gene therapy suppresses Twist mRNA (G and H) and protein (I) expression in obstructed kidney. Representative RT-PCR (G) and Western blot (I) results and quantitative data (H) are presented. **P Ͻ 0.01 versus sham controls; †P Ͻ 0.05 versus pcDNA3 (n ϭ 4to5).

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canonical and noncanonical pathways of Wnt signaling are at work, ensuring a functional multiplicity and signal output homeostasis under normal circum- stances. It is becoming apparent that the relative silence of Wnt/␤-catenin signal- ing in adult kidney does not represent a lack of Wnt expression but might in fact result from a balance of the expression between the stimulatory and inhibitory Wnt and Fzd genes,28 as well as the con- stitutive expression of various Wnt an- tagonists. One of the striking observations in this study is the concurrent induction of mul- tiple Wnt genes in the fibrotic kidney af- ter obstructive injury. In fact, except for Wnt5b, Wnt8b, and Wnt9b, all members of Wnt family genes were upregulated, al- beit divergent in induction dynamics (Figure 1). In addition, the expression of numerous Fzd receptor genes was in- duced (Figure 3). Perhaps unexpected, there is no single gene among both Wnt and Fzd receptor families whose expres- Figure 9. Blockade of Wnt signaling by DKK1 inhibits ␣-SMA and FSP-1 expression sion was suppressed during the entire ex- in the obstructed kidney. (A and B) Representative RT-PCR analysis (A) and quan- perimental period. This suggests that titative data (B) show that delivery of DKK1 gene suppressed renal ␣-SMA mRNA most members of Wnt family proteins are expression after obstructive injury. (C and D) Representative Western blot (C) and responsive positively to injurious stimuli quantitative data (D) demonstrate that DKK1 suppressed renal ␣-SMA protein after UUO. Although such a reexpression expression after obstructive injury. Numbers (1, 2, and 3) denote each individual or induction of the embryonic genes after animal in a given group. Relative ␣-SMA mRNA (B) or protein (D) levels (fold injury is not without precedent, the ob- induction over sham controls) were reported after normalization with GAPDH, servation that 16 of 19 Wnt genes were Ͻ † Ͻ ϭ respectively. **P 0.01 versus sham controls; P 0.05 versus pcDNA3 (n 4to induced concurrently in a particular set- ␣ 5). (E) Representative micrographs demonstrate -SMA protein localization by ting of disease is astonishing. Naturally, immunofluorescence staining and FSP-1 protein by immunohistochemical staining induction of Wnt and Fzd genes would in different groups as indicated. Bar ϭ 40 ␮m. (F) Quantitative determination of Ͻ lead to the stabilization and accumula- FSP-1 expression in different groups as indicated. **P 0.01 versus sham controls; ␤ †P Ͻ 0.05 versus pcDNA3 (n ϭ 4to5). tion of -catenin, the mediator of the Wnt canonical pathway,29 by preventing in which the Wnt/Ca2ϩ and Wnt/planar cell polarity (PCP) its phosphorylation and degradation via ␤-transducin re- ϩ routes are best described.8 In the Wnt/Ca2 pathway, Wnts peat-containing protein–mediated ubiquitination.1,5 In- bind to Fzd receptor to activate Dvl; however, this results in deed, despite that Wnt ant-agonists DKK1 through 4 were an increase in intracellular Ca2ϩ and activation of protein slightly induced (Figure 4), ␤-catenin protein is markedly kinase C and calmodulin kinase II.23,24 Functionally, activa- increased and predominantly localized in the cytoplasm and tion of the Wnt/Ca2ϩ pathway may antagonize the Wnt/␤- nuclei of tubular epithelial cells (Figure 5), indicative of a catenin signaling, providing a negative feedback mecha- prevailed Wnt/␤-catenin signaling in diseased kidney. nism.25 The Wnt/PCP pathway uses Fzd and Dvl as well but It should be pointed out that we do not know the sources does not lead to ␤-catenin stabilization or Ca2ϩ influx in and localization of Wnts in vivo in this study, because of the regulating the generation of PCP.26,27 The noncanonical lack of specific and workable antibodies against many verte- pathways are often operated in response to distinct group of brate Wnt proteins in immunohistochemical studies. A previ- Wnt ligands and Fzd receptors, such as Wnt4, Wnt5a, ous in situ hybridization study showed that Wnt4 mRNA ex- Wnt11, and Fzd2 through 410,23; therefore, although the role pression was induced in collecting duct epithelium and in of specific Wnts and Fzd receptors in normal kidney re- activated interstitial myofibroblasts after various injuries.18 mains to be determined, in view of the expression pattern of Because Wnt is a secreted protein, cells may readily access and different Wnts and Fzd receptor, we can speculate that both respond to Wnts in the extracellular mellitus in an autocrine or

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duces the de novo expression of mesen- chymal markers.34,35 In view of the im- portance of tubular EMT in the pathogenesis of renal fibrosis,36,37 Twist may function as a critical mediator of Wnt/␤-catenin in promoting EMT and renal fibrogenesis. Of note, Wnt/␤-cate- nin signaling may be amplified in dis- eased kidney because one of its targets is LEF1,31 the DNA-binding transcription factor that interacts with ␤-catenin, lead- ing to the formation of a trans-activating protein complex. As another direct target of Wnt/␤-catenin,33 the significance of an increased fibronectin expression in the evolution of renal fibrosis can be easily envisioned. Notably, the expression of c- Myc, a widely known target of Wnt/␤- catenin that is implicated in regulating cell proliferation,1,3,32 is not exactly cor- related with ␤-catenin levels (Figure 6). Figure 10. Inhibition of Wnt signaling by DKK1 reduces renal interstitial injury and The reason behind this discrepancy is un- total collagen deposition after UUO. (A through F) Kidney sections were stained with known, but it could suggest that a certain Picrosirius red (A through C) and Masson trichrome (D through F) for assessment of collagen deposition. Representative micrographs from different groups as indicated group of Wnts, such as Wnt1, Wnt7a, and are shown. (A and D) Sham controls. (B and E) Mice that underwent UUO and were Wnt7b, which share a similar expression administered an injection of pcDNA3. (C and F) Mice that underwent UUO and were pattern with c-Myc after UUO, might be administered an injection of pFlag-DKK1. Bar ϭ 40 ␮m. (G) Graphic presentation responsible specifically for c-Myc induc- shows renal interstitial volume in different groups as indicated. (H) DKK1 treatment tion in diseased kidney. It should be reduced tissue hydroxyproline content in the obstructed kidney. Tissue hydroxyproline noted that we cannot exclude the possi- was expressed ␮g/mg dry kidney weight. **P Ͻ 0.01 versus sham controls; †P Ͻ 0.05 bility that other transcription factors may versus pcDNA3 (n ϭ 4to5). also participate in the regulation of Wnt target genes in fibrotic kidney in vivo. paracrine manner, regardless of the sources. Because tubular More studies are clearly needed in the area. epithelial cells express the majority of Fzd receptors in vitro Our study also suggests that targeting Wnt/␤-catenin sig- (data not shown), it is plausible to assume that they could be naling might be an effective strategy to hinder the progression the main targets of Wnt signaling in diseased kidney. This no- of renal interstitial fibrosis. Delivery of exogenous DKK1 via tion is supported by the observation that ␤-catenin was pri- naked plasmid injection led to substantial expression of exog- marily activated in tubular epithelium after obstructive injury enous DKK1 in kidney as well as in liver (Figure 7). Because it (Figure 5). is a secreted protein, exogenous DKK1 produced in kidney in The ultimate responses of Wnt/␤-catenin signaling situ and/or from liver via circulation presumably can access to ought to reflect on regulating particular gene expression. In and target the hyperactive Wnt signaling in diseased kidney. that regard, numerous target genes downstream of Wnt/␤- This results in a reduction of ␤-catenin accumulation and sup- catenin have been characterized in diverse types of cells. On pression of Wnt/␤-catenin target genes in fibrotic kidney, lead- the basis of previous reports in other biologic systems,30–33 ing to a reduction of renal matrix deposition and fibrosis. This we identified several putative target genes of Wnt/␤-catenin conclusion is also substantiated by a previous report demon- in fibrotic kidney, including Twist, LEF1, fibronectin, and strating that administration of recombinant sFRP4 protein was c-Myc. The expression of these genes is closely correlated able to ameliorate renal fibrotic lesions.17 It is worthwhile to with ␤-catenin abundance in vivo (Figure 6) and is inhibited stress that DKK proteins as Wnt antagonists are unique be- specifically after Wnt signaling is blocked with DKK1 (Fig- cause they specifically block Wnt ligands binding to LRP5/6, ures 8 and 11). Among these genes, Twist, a transcription the co-receptors that are obligatory for transmitting the ca- factor of the basic helix-loop-helix class, has been shown to nonical pathway of Wnt/␤-catenin signaling11,12; therefore, play a pivotal role in mediating epithelial-to-mesenchymal our observation that DKK1 inhibits renal fibrosis clearly un- transition (EMT) in different circumstances.30 Twist not derscores a pivotal role of the canonical pathway of a hyperac- only is able to repress E-cadherin gene transcription by tive Wnt/␤-catenin signaling in the pathogenesis of renal in- binding to the E-boxes in its promoter region but also in- terstitial fibrosis.

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RT-PCR Total RNA was prepared from kidney tissue by using TRIzol reagent, according to the protocol specified by the manufacturer (Invitrogen, Carlsbad, CA). After reverse transcription of the RNA, cDNA was used as a template in PCR re- actions using gene-specific primer pairs. Ap- proximately 25 to 30 cycles for amplification in the linear range were used. Some experiments were performed without addition of RT. After quantification of band intensities by using den- sitometry, the relative steady-state levels of mRNA were calculated after normalizing to ␤-actin or glyceraldehyde-3-phosphate dehy- drogenase. The sequences of the primer sets are given in the Supplemental Table 3.

Western Blot Analysis The preparation of kidney tissue homogenates and Western blot analysis of protein expression were carried out by using routine procedures as described previously.39 The primary antibodies were obtained from the following sources: Anti- Figure 11. Blockade of Wnt signaling by DKK1 inhibits type I collagen and fibronectin Wnt4 (AF475; R&D Systems, Minneapolis, expression in obstructive nephropathy. (A and C) Representative RT-PCR analysis of MN); anti-Wnt7a (sc-26361), anti–c-Myc (sc- renal mRNA levels of type I collagen (A) and fibronectin (C) in different treatment 764), and anti-Twist (sc-15393; Santa Cruz Bio- groups as indicated. Numbers (1, 2, and 3) denote each individual animal in a given ␤ group. (B and D) Graphic presentation of the mRNA levels of type I collagen (B) and technology, Santa Cruz, CA); anti– -catenin fibronectin (D) in different groups. Relative mRNA levels were calculated and are (cat. no. 610154; BD Transduction Laboratories, expressed as fold induction over sham controls (value ϭ 1.0) after normalization with San Jose, CA); anti-Flag (M2; F3165) and anti– ␤-actin. **P Ͻ 0.01 versus sham controls; †P Ͻ 0.05 versus pcDNA3 (n ϭ 4 to 5). (E) ␣-SMA (clone 1A4) (Sigma); and anti–glyceral- Representative micrographs demonstrate type I collagen and fibronectin localization dehyde-3-phosphate dehydrogenase (Ambion, by immunofluorescence staining in different groups as indicated. Bar ϭ 40 ␮m. Austin, TX). CONCISE METHODS Immunohistochemical and Immunofluorescence Animals Staining Male CD-1 mice that weighed approximately 18 to 22 g were pur- Immunohistochemical staining of kidney sections was performed by chased from Harlan Sprague Dawley (Indianapolis, IN). UUO was an established protocol.40 Paraffin-embedded sections were stained performed using an established protocol, as described previously.38 with polyclonal rabbit anti–␤-catenin antibody (ab-15180; Abcam, Sham-operated mice were used as normal controls. At different time Cambridge, MA) and polyclonal rabbit anti-S100A4 (FSP-1) anti- points after surgery, groups of mice (n ϭ 5) were killed and kidneys body (A5114; DakoCytomation, Glostrup, Denmark) using the Vec- were removed for various analyses. For delivery of human DKK1 tor M.O.M. immunodetection kit, according to the protocol specified gene, naked DKK1 expression plasmid (pFlag-DKK1; provided by Dr. by the manufacturer (Vector Laboratories, Burlingame, CA). Indirect Xi He, Harvard Medical School, Boston, MA)11 was injected intrave- immunofluorescence staining was carried out according to estab- nously at 1 mg/kg body wt before (day Ϫ1) UUO, by use of a hydro- lished procedures.39 Briefly, kidney cryosections were incubated with dynamics-based in vivo gene transfer approach, as described previ- specific primary antibodies against ␣-SMA, collagen I (cat. no. 1310- ously.20,21 Control UUO mice were administered an injection of 01; Southern Biotech, Birmingham, AL), and fibronectin (cat. no. empty vector pcDNA3 plasmid in an identical manner. Mouse model 610078; BD Transduction Laboratories), respectively, followed by of adriamycin nephropathy was established according to the protocol staining with cyanine Cy3-conjugated secondary antibody (Jackson described previously.19 Briefly, male BALB/c mice (Harlan Sprague- ImmunoResearch Laboratories, West Grove, PA). Slides were viewed Dawley) were administered via tail vein an injection of adriamycin with a Nikon Eclipse E600 microscope equipped with a digital camera (doxorubicin hydrochloride; Sigma, St. Louis, MO) at 10 mg/kg body (Melville, NY). Nonimmune normal control IgG was used to replace wt. Mice were killed at 5 wk after adriamycin injection, and kidney the primary antibody as negative control, and no staining occurred. tissue was collected for various analyses. Animal protocols were ap- Quantification of the staining was carried out by a grid counting– proved by the Institutional Animal Care and Use Committee at the based, computer-aided morphometric analysis, as described previ- University of Pittsburgh. ously.41

774 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 765–776, 2009 www.jasn.org BASIC RESEARCH

Quantitative Determination of DKK1 Protein Levels ACKNOWLEDGMENTS DKK1 protein levels in plasma and tissues were determined quantita- tively by an ELISA using a specific DKK1 detection kit (R&D Sys- This work was supported by the National Institutes of Health grants tems). This ELISA kit detects both human (exogenous) and mouse DK061408, DK064005, and DK071040. endogenous DKK1 protein. Plasma samples were collected from mice that were administered an injection of either pFlag-DKK1 expression DISCLOSURES vector or pcDNA3 plasmid. For determining tissue DKK1 levels, liver None. and kidney from mice were homogenized in the extraction buffer containing 20 mM Tris HCl (pH 7.5), 2 M NaCl, 0.1% Tween-80, 1 mM EDTA, and 1 mM PMSF, as described previously.42 After centrif- REFERENCES ugation at 19,000 ϫ g for 20 min at 4°C, the supernatant was recov- ered for DKK1 assay, according to the protocol specified by the man- 1. 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