Blockade of Wnt/ß-Catenin Signaling by Paricalcitol Ameliorates

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Blockade of Wnt/␤-Catenin Signaling by Paricalcitol Ameliorates Proteinuria and Kidney Injury

Weichun He, Young Sun Kang, Chunsun Dai, and Youhua Liu

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

ABSTRACT Recent studies implicate Wnt/␤-catenin signaling in podocyte dysfunction. Because vitamin D analogs can inhibit ␤-catenin in other tissues, we tested whether the vitamin D analog paricalcitol could ameliorate podocyte injury, proteinuria, and renal fibrosis in adriamycin (ADR) nephropathy. Compared with vehicle-treated controls, paricalcitol preserved expression of nephrin, podocin, and WT1; prevented proteinuria; and reduced glomerulosclerotic lesions induced by ADR. Paricalcitol also inhibited expression of proinflammatory cytokines, reduced renal infiltration of monocytes/macrophages, hampered activation of renal myofibroblasts, and suppressed expression of the fibrogenic TGF-␤1, CTGF, fibronectin, and types I and III collagen. Selective suppression of renal Wnt4, Wnt7a, Wnt7b, and Wnt10a expression after ADR accompanied these renoprotective effects of paricalcitol. Significant upregulation of ␤-catenin, predominantly in podocytes and tubular epithelial cells, accompanied renal injury; paricalcitol largely abolished this induction of renal ␤-catenin and inhibited renal expression of Snail, a downstream effector of Wnt/␤-catenin signaling. Administration of paricalcitol also ameliorated established proteinuria. In vitro, paricalcitol induced a physical interaction between the vitamin D receptor and ␤-catenin in podocytes, which led to suppression of ␤-catenin–mediated gene transcription. In summary, these findings suggest that paricalcitol prevents podocyte dysfunction, proteinuria, and kidney injury in adriamycin nephropathy by inhibiting Wnt/␤-catenin signaling.

J Am Soc Nephrol 22: 90–103, 2011. doi: 10.1681/ASN.2009121236

Proteinuria is an early and predominant pathologic oping a strategy aimed to target the Wnt/␤-catenin feature of a wide variety of primary glomerular dis- signal pathway may be a plausible approach for the eases that progress to end-stage renal failure. In- treatment of proteinuric kidney disorders. creasing evidence suggests that podocyte injury is Wnt/␤-catenin is an evolutionarily conserved one of the major causes leading to defective glomer- cellular signaling system that plays an essential role ular filtration, which results in proteinuria.1–3 It has in diverse array of biologic processes such as orga- been well documented that proteinuria not only is a nogenesis, tissue homeostasis, and pathogenesis of marker for the progression of chronic kidney dis- many human diseases.8,9 Aberrant regulation of eases (CKD) but also acts as a pathogenic mediator Wnt/␤-catenin has been implicated in many types that incites renal inflammation and promotes tubu- of kidney diseases including obstructive nephropa- lar injury and interstitial fibrosis.4,5 Despite the fact that the importance of podocyte injury in protein- Received December 10, 2009. Accepted August 18, 2010. uria is well recognized, the mechanisms and signal pathways leading to podocyte damage in the vast Published online ahead of print. Publication date available at www.jasn.org. majority of proteinuric kidney disorders remain Correspondence: Dr. Youhua Liu, Department of Pathology, Uni- poorly understood. We have recently shown that versity of Pittsburgh School of Medicine, S-405 Biomedical Sci- Wnt/␤-catenin signaling plays a critical role in pro- ence Tower, 200 Lothrop Street, Pittsburgh, PA 15261. Phone: moting podocyte injury, proteinuria, and renal fi- 412-648-8253; Fax: 412-648-1916; E-mail: [email protected] brosis.6,7 In this context, it is conceivable that devel- Copyright © 2011 by the American Society of Nephrology

90 ISSN : 1046-6673/2201-90 J Am Soc Nephrol 22: 90–103, 2011 www.jasn.org BASIC RESEARCH thy, chronic allograft nephropathy, diabetic nephropathy, markedly elevated at 5 weeks after ADR injection, and admin- polycystic kidney disease, focal and segmental glomeruloscle- istration of paricalcitol largely prevented proteinuria in this rosis, and adriamycin nephropathy.6,7,10–12 Wnt proteins model. Kidney histology by Masson-trichrome staining re- transmit their signal across the plasma membrane through in- vealed clearly visible nephropathy at 5 weeks after ADR injec- teracting with the Frizzled (Fzd) receptors, as well as their co- tion, characterized by the fibrotic lesions in the glomeruli (Fig- receptors, members of the LDL receptor-related protein 5/6. ure 1B, arrow), tubular dilation with proteinous fluid in the Upon binding to their receptors/coreceptors, Wnt proteins in- lumens (Figure 1B, asterisks), as well as expanded interstitial duce a series of downstream signaling events, resulting in space. Consistent with the proteinuria data, administration of ␤-catenin dephosphorylation and stabilization. This allows paricalcitol ameliorated kidney injury after ADR injection ␤-catenin to translocate into the nuclei, wherein it binds to T (Figure 1B). Quantitative determination of kidney fibrotic le- cell factor (TCF)/lymphoid enhancer-binding factor to stimu- sion among different groups at 5 weeks after ADR injection is late the transcription of Wnt target genes.13 On the basis of this presented in Figure 1C. To assess more acute effects of parical- canonical pathway of Wnt signaling, it is conceivable that ei- citol on the development of proteinuria, another set, short du- ther inhibiting Wnt expression or repressing ␤-catenin tran- ration of animal experiments was performed. As shown in Fig- scriptional activity could be an effective way to control the ure 1D, robust albuminuria was evident in mice at 7 days after Wnt/␤-catenin signaling. ADR injection, and paricalcitol also significantly reduced uri- Earlier studies indicate that vitamin D analogs are able to nary albumin level in this setting. promote the differentiation of colon carcinoma cells by inhibiting ␤-catenin signaling.14 This action of vitamin D appears to Paricalcitol Prevents Podocyte Injury and Reduces be mediated by ligand-activated vitamin D receptor (VDR) Glomerular Lesions In Vivo competing with transcription factor TCF-4 for ␤-catenin Because podocyte injury is an early and predominant patho- binding. These observations suggest that vitamin D and its logic feature of this model,6,25 we next investigated the effects receptor compose an endogenous negative regulator that of paricalcitol on podocyte damage and glomerular lesions in tightly controls ␤-catenin signaling.15,16 Interestingly, defi- vivo. As shown in Figure 2A, comparing with normal controls, ciency in vitamin D and its active metabolites is highly preva- podocyte slit diaphragm–associated proteins nephrin and lent in advanced stage CKD,17,18 in which Wnt/␤-catenin sig- podocin were substantially down-regulated in the kidney at 5 naling is activated.7,11,12 Consistently, administration of weeks after ADR injection, as illustrated by immunofluores- vitamin D analogs are able to reduce proteinuria and promote cence staining. Western blot analyses of the isolated glomeruli overall survival in patients with CKD by a mechanism that is from different groups of mice produced similar results (Figure independent of serum parathyroid hormone, phosphorus, and 2B). However, these slit diaphragm–associated proteins were calcium levels.19–24 Taken together, these results led us to hy- restored after paricalcitol treatment (Figure 2, A and B), indi- pothesize that administration of vitamin D analog might be cating an effective preservation of podocyte integrity. able to effectively prevent podocyte dysfunction, proteinuria, We also examined the expression of Wilms tumor 1 (WT1) and kidney injury by modulating Wnt/␤-catenin signaling. protein, a pivotal transcription factor that is essential for the Here we examined the therapeutic effects of paricalcitol maintenance of the differentiated features of adult podo- (19-nor-1,25-hydroxy-vitamin D2), a synthetic and active vi- cytes.27,28 As illustrated in Figure 2 (A and B), WT1 protein tamin D analog, in adriamycin (ADR) nephropathy. Our data expression was also markedly suppressed in the glomeruli after demonstrate that paricalcitol mitigates proteinuria and kidney ADR injury, and paricalcitol treatment restored WT1 protein injury by inhibiting Wnt/␤-catenin signaling. These studies expression. Of note, despite a significant decrease in the num- indicate that blocking Wnt/␤-catenin signaling is a plausible bers of the WT1-positive cells, podocyte apoptosis as shown by strategy for therapeutic intervention of proteinuric kidney dis- terminal deoxynucleotidyl transferase-mediated dUTP nick- orders. end labeling staining was extremely rare (Ͻ2 per 100 glomerular cross-sections) at 5 weeks after ADR injection (data not shown). RESULTS Figure 2 (C through E) shows that podocyte injury and glomerular lesions were an early event in this model. At 7 days Paricalcitol Ameliorates Proteinuria and Kidney Injury after ADR injection, nephrin, podocin, and WT1 were already in Adriamycin Nephropathy down-regulated, and paricalcitol was able to largely preserve We investigated the effects of paricalcitol on ADR nephropa- their expression (Figure 2, C through E). thy, a model characterized by initial podocyte injury and albuminuria and subsequent renal inflammation and fibrosis.25,26 Paricalcitol Inhibits Renal Inflammation Of interest, three of eight mice with severe proteinuria in ADR We next examined the effects of paricalcitol on renal inflam- group died between 3 and 5 weeks after ADR injection, mation at 5 weeks after ADR injection, because an increased whereas all eight mice survived in the ADR group receiving renal infiltration of inflammatory cells is a pathologic feature paricalcitol. As shown in Figure 1A, urinary albumin levels of this model. To this end, we initially investigated the expres-

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Figure 1. Paricalcitol ameliorates proteinuria and kidney injury in adriamycin nephropathy. (A) SDS-PAGE analysis shows the abundance and composition of urinary proteins in different groups of mice at 5 weeks after ADR injection. Urine samples after normalization to creatinine were analyzed on SDS-PAGE, with BSA (1 ␮g) loaded on the adjacent lane. The numbers (1 and 2) indicate each individual, representative animal in a given group. (B) Representative micrographs demonstrate kidney injury at 5 weeks after ADR injection in different groups of mice as indicated. Kidney sections were subjected to Masson-trichrome staining. The asterisks indicate the dilated tubules with proteinous fluid in the lumens. The arrows indicate sclerotic glomeruli. Images with different magnifications were shown. The scale bar in the top panels indicates 250 ␮m; that in the bottom panels indicates 50 ␮m. (C) Quantitative determination of kidney fibrotic lesions in different groups at 5 weeks after ADR injection. **P Ͻ 0.01 versus normal controls; †P Ͻ 0.05 versus ADR alone (n ϭ 5 to 8). (D) Urinary albumin levels in mice at 7 days after ADR injection. Urinary albumin was expressed as mg/mg creatinine. **P Ͻ 0.01 versus normal controls; †P Ͻ 0.05 versus ADR alone (n ϭ 6). CTL, control; Pari., paricalcitol. sion of several proinflammatory cytokines including the Paricalcitol Reduces Renal Fibrotic Lesions after regulated on activation normal T cell expressed and secreted Adriamycin Injury (RANTES), also known as CC-chemokine ligand 5, TNF-␣, Because ADR injury inevitably leads to renal fibrotic lesions, and monocyte chemotactic protein-1 (MCP-1), also known as we next examined the effects of paricalcitol on renal fibrosis in CC-chemokine ligand 2. As shown in Figure 3A, at 5 weeks this model. To this end, we initially investigated the expression after ADR injection, the renal mRNA levels for RANTES, of TGF-␤1 and connective tissue growth factor (CTGF), two TNF-␣, and MCP-1 were markedly up-regulated. Administra- major fibrogenic cytokines that are involved in the pathogen- tion of paricalcitol substantially inhibited renal expression esis of a wide array of CKD. As shown in Figure 4 (A through of these proinflammatory cytokines (Figure 3, B and C), as C), real-time RT-PCR analyses demonstrated that both determined by quantitative real-time reverse transcriptase TGF-␤1 and CTGF mRNA levels were increased in the kidney (RT)-PCR approach. Consistently, immunohistochemical at 5 weeks after ADR injection, and paricalcitol significantly staining for F4/80 antigen, a marker for myeloid cells in- abrogated their induction. Analyses of the expression of several cluding monocytes/macrophages and dendritic cells, showed interstitial matrix genes such as fibronectin and type I and type that an increased renal infiltration of the F4/80-positive cells in III collagen in different groups of mice also indicated that pari- kidney parenchyma after ADR injection (Figure 3, D and E). calcitol was able to inhibit the mRNA expression of major in- Notably, virtually all F4/80-positive cells were found in the terstitial matrix genes induced by ADR (Figure 4, D through interstitium but not in the glomeruli (Figure 3E). Paricalcitol G). These results are consistent with and supported by an al- effectively blocked renal infiltration of these F4/80-positive in- tered renal collagen deposition revealed by Masson-trichrome flammatory cells (Figure 3F). staining (Figure 1, B and C).

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Figure 2. Paricalcitol preserves nephrin, podocin, and WT1 expression and prevents podocyte injury in vivo. (A and C) Representative micrographs show the abundance and distribution of nephrin, podocin, and WT1 proteins in the glomeruli of different groups of mice as indicated at 5 weeks (A) or 1 week (C) after ADR injection, respectively. Scale bar, 20 ␮m. (B and D) Western blot analyses demonstrate that paricalcitol preserved nephrin, podocin, and WT1 expression at 5 weeks (B) or 1 week (D) after ADR injection, respectively. Glomerular lysates from different groups of mice were immunoblotted with specific antibodies against nephrin, podocin, WT1, and actin, respectively. The numbers (1, 2, and 3) indicate each individual glomerular preparation isolated from a pool of two animals. (E) Quantitative determination of the relative abundances of nephrin, podocin, and WT1 in different groups at 1 week after ADR injection. *P Ͻ 0.05 versus normal controls; †P Ͻ 0.05 versus ADR alone (n ϭ 3).

We further examined renal expression of ␣-smooth muscle able to selectively inhibit specific Wnt expression induced after actin (␣-SMA) and myofibroblast activation in different ADR injury. groups of mice. As shown in Figure 4 (H and I), renal ␣-SMA We also examined the expression of the members of the protein levels were dramatically increased after ADR injury, Dickkopf (DKK) family of endogenous Wnt antagonists in dif- suggesting myofibroblast activation in this model. This induc- ferent groups of mice. As shown in Figure 5 (D and E), al- tion of renal ␣-SMA, however, was largely abolished by pari- though ADR injury also caused an induction of these DKK calcitol (Figure 4, H and I). Similar results were obtained when genes, paricalcitol did not significantly affect their expression examining myofibroblast activation by immunofluorescence after ADR administration. staining for ␣-SMA protein (data not shown). Paricalcitol Blocks ␤-Catenin Activation and Paricalcitol Represses Renal Expression of Wnt Genes Suppresses Its Downstream Snail Expression To provide mechanistic insights into the renal protective effi- Because ␤-catenin is the principal mediator of the canonical cacy of paricalcitol in ADR nephropathy, we investigated its Wnt signaling, we next examined its regulation in ADR ne- effects on the activation of Wnt/␤-catenin signaling, because phropathy. As shown in Figure 6 (A and B), Western blot anal- recent studies suggest a critical role of this signal pathway in yses revealed a dramatic increase in renal ␤-catenin protein podocyte dysfunction and renal fibrosis.6,7 A comprehensive abundance at 5 weeks after ADR injection. Quantitative deter- analysis of all 19 Wnt genes has demonstrated that numerous mination showed a more than 150-fold induction of ␤-catenin Wnts were up-regulated in the kidney in this model, as re- protein over the controls in this model (Figure 6B). Immuno- ported previously.7 We found that paricalcitol could specifi- histochemical staining demonstrated that ␤-catenin was pre- cally inhibit renal expression of multiple Wnts, including dominantly localized at renal tubular epithelial cells and glo- Wnt4, Wnt7a, Wnt7b, and Wnt10a (Figure 5, A through C). merular podocytes (Figure 6, D and F), whereas the staining for However, paricalcitol appeared not to suppress Wnt3 expres- ␤-catenin in normal kidney was weak (Figure 6C). Cytoplas- sion; rather, it slightly induced its expression in this model mic and nuclear staining of ␤-catenin was clearly visible in (Figure 5, A and C). These results suggest that paricalcitol is glomerular podocytes (Figure 6F, yellow arrowheads), as well

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Figure 3. Paricalcitol inhibits proinflammatory cytokines expression and reduces renal infiltration of monocytes/macrophages. (A) Representative RT-PCR results show renal mRNA expression of RANTES, TNF-␣, and MCP-1 at 5 weeks after ADR injection in different groups of mice as indicated. The numbers (1, 2, and 3) denote each individual animal in a given group. (B and C) Graphic presentation shows the relative mRNA levels of RANTES, TNF-␣ (B), and MCP-1 mRNA levels (C) determined by quantitative, real-time RT-PCR in different groups. Relative mRNA levels were determined after normalization with ␤-actin and expressed as fold induction over controls. The data are expressed as the means Ϯ SEM (n ϭ 5 to 8). **P Ͻ 0.01 versus normal controls. †P Ͻ 0.05 versus ADR alone. (D through F) Representative micrographs show renal infiltration of F4/80-positive myeloid cells including monocytes/macrophages and dendritic cells at 5 weeks after ADR injection in different groups of mice as indicated. The arrows indicate F4/80-positive cells. (D) Normal control. (E) ADR alone. (F) ADR plus paricalcitol. Scale bar, 50 ␮m. CTL, control; Pari., paricalcitol; g, glomeruli. as in renal tubular epithelia, suggesting the activation of this uria was significantly reduced at 7 days after ADR when the signaling in these cells. Of interest, administration of parical- preventive protocol was used (group 3). Interestingly, urinary citol largely prevented ␤-catenin induction and activation albumin levels also started to decline, albeit not statistically (Figure 6, A, B and E). significantly (P ϭ 0.155, n ϭ 6), in just 1 day after paricalcitol We further examined the expression of Snail, a downstream administration starting at 6 days after ADR (group 5) com- mediator of the Wnt/␤-catenin signaling in podocytes.6 As pared with ADR alone (group 2). At 14 and 21 days after ADR, shown in Figure 7 (A and B), Snail protein was markedly in- albuminuria was significantly reduced in all three groups that duced in the injured kidney after ADR injection; and adminis- received paricalcitol, regardless of the starting time points tration of paricalcitol largely blocked renal Snail induction. when paricalcitol administration was initiated (Figure 8B). In Similarly, renal Snail mRNA was also induced, to a lesser ex- fact, the levels of urinary albumin in these paricalcitol-treated tent, after ADR injury, which was blocked by paricalcitol (Fig- groups were indistinguishable. Notably, a time-dependent re- ure 7, C and D). Figure 7E shows the putative signaling path- gression of proteinuria was observed in all three groups that ways leading to Snail induction by ADR. These results suggest received paricalcitol (Figure 8B). Analyses of urinary proteins that paricalcitol is able to target a key pathogenic signaling by by SDS-PAGE revealed the similar results (Figure 8C), suggest- inhibiting Wnt/␤-catenin and its downstream Snail. ing that paricalcitol is able to ameliorate and reverse an established proteinuria. Consistently, renal histology showed that Paricalcitol Reverses an Established Proteinuria significant morphologic lesions were evident in the kidney at 3 We next investigated whether delayed administration of pari- weeks after ADR injection (Figure 8D, panel b), and these his- calcitol is still effective in ameliorating proteinuria, a scenario tologic injuries were markedly mitigated in all three groups that is obviously of clinical relevance. As depicted in Figure 8A, that received paricalcitol (Figure 8D, panels c through e). several different treatment protocols were used. In the preven- We further examined renal ␤-catenin expression in differ- tive protocol (group 3), paricalcitol was commenced 1 day be- ent groups. As shown in Figure 8 (E and F), ␤-catenin abun- fore ADR injection. The mice in groups 4 and 5 were given dance in the injured kidney at 3 weeks after ADR was increased paricalcitol starting at either 2 days after ADR, a time point approximately 45-fold over the controls. Treatment with pari- when albuminuria is just about to emerge, or 6 days after ADR, calcitol starting at 1 day before or 2 days after ADR, respec- a time point when robust albuminuria is already established in tively, significantly prevented renal ␤-catenin induction. How- this model,6,29 respectively. As shown in Figure 8B, albumin- ever, delayed administration of paricalcitol at 6 days after ADR

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Figure 4. Paricalcitol inhibits renal expression of TGF-␤1, CTGF, and matrix genes and reduces myofibroblast activation after ADR injury. (A through C) Representative RT-PCR results (A) and graphic presentation (B and C) showed the mRNA expression of profibrotic cytokines TGF-␤1 and CTGF in different groups of mice as indicated at 5 weeks after ADR injection. The numbers (1, 2, and 3) denote each individual animal in a given group. (D through G) Representative RT-PCR (D) and graphic presentation of the mRNA levels of interstitial matrix genes fibronectin (E), type I (F), and type III collagen (G) in different groups of mice. The relative mRNA levels were determined by quantitative real-time RT-PCR analysis, calculated after normalization with ␤-actin, and expressed as fold induction over normal controls. (H and I) Western blot analyses show the ␣-SMA protein expression in different groups of mice as indicated at 5 weeks after ADR injection. Representative Western blots (H) and quantitative determination of ␣-SMA protein levels (I) are presented. The data are expressed as the means Ϯ SEM (n ϭ 5 to 8). *P Ͻ 0.05, **P Ͻ 0.01 versus normal controls; †P Ͻ 0.05, ††P Ͻ 0.01 versus ADR alone. CTL, control; Pari., paricalcitol; Veh., vehicle. was clearly less effective in inhibiting ␤-catenin expression cubation of podocytes with paricalcitol induced VDR nuclear (Figure 8, E and F). This discrepancy between renal ␤-catenin translocation (Figure 9A). However, it appeared that pretreat- abundance (Figure 8, E and F) and the severity of albuminuria ment with paricalcitol did not block ␤-catenin nuclear trans- and histologic lesions (Figure 8, B through D) in three parical- location triggered by ADR in podocytes (Figure 9A). Of note, citol-treated groups raise the possibility that paricalcitol may neither paricalcitol nor ADR affect total cellular levels of VDR also directly disrupt ␤-catenin signaling, in addition to inhib- and ␤-catenin after a short period of incubation (Figure 9B). iting Wnt expression and ␤-catenin accumulation. Given that both VDR and ␤-catenin undergo nuclear translocation after stimulation, we next tested whether activated VDR Paricalcitol Induces VDR to Interact with ␤-Catenin interacts with nuclear ␤-catenin by using a coimmunoprecipi- and Sequestrate Its Transcription Activity tation approach. As shown in Figure 9 (C and D), incubation of To explore whether paricalcitol can directly affect ␤-catenin– mouse podocytes with paricalcitol induced VDR to interact mediated signaling, we used in vitro cultured mouse podocytes with ␤-catenin, as shown by increased VDR/␤-catenin com- as a model system. We first examined whether paricalcitol plex formation after paricalcitol stimulation. We further as- blocked ␤-catenin nuclear translocation, an obligatory step for sessed the functional consequence of VDR/␤-catenin interac- ␤-catenin to control its target gene transcription in the nu- tion by examining the ␤-catenin–mediated gene transcription cleus. As shown in Figure 9A, treatment of mouse podocytes in a luciferase reporter system. As shown in Figure 8E, parical- with ADR induced ␤-catenin activation and its nuclear trans- citol could significantly repress ␤-catenin–mediated gene tran- location, as nuclear ␤-catenin level was induced. Similarly, in- scription in cultured podocytes. Similarly, ADR induced

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tion (Figure 9H). These studies underscore that vitamin D is a potent endogenous, nat- ural antagonist of Wnt/␤-catenin signaling in vivo. Our results also indicate that targeting this signaling could be an effective way to mitigate proteinuria and kidney injury in a variety of pathologic conditions. Given the inherent nature of ADR nephropathy, our attention in this study is primarily focused on the ability of paricalcitol to mitigate podocyte dysfunction, proteinuria, and glomerular lesions. Albu- minuria, as well as podocyte foot process effacement, typically occurs at 3 days and becomes prominent at 6 days after ADR injection,6,29 whereas significant inflammation and tubulointerstitial lesions are not seen in this early stage. In considering the pathologic sequences of this model, it is Figure 5. The expression of Wnt genes is selectively inhibited by paricalcitol in the conceivable that the reno-protective effect kidney after ADR injury. (A) Representative RT-PCR results show renal mRNA expres- of paricalcitol may be primarily attribut- sion of various Wnt genes in different groups of mice as indicated at 5 weeks after ADR injection. The numbers (1, 2, and 3) denote each individual animal in a given group. able to its prevention of podocyte injury. (B and C) Graphic presentation of various Wnts mRNA levels in different groups. This notion is further substantiated by the Relative mRNA levels were determined after normalization with ␤-actin and expressed observations that paricalcitol prevents the as fold induction over normal controls. (D and E) RT-PCR results show renal expression loss of podocyte-specific nephrin, podocin, of various DKK genes in different groups of mice as indicated. Relative mRNA levels and WT1 as early as 7 days after ADR injec- were determined after normalization with ␤-actin and expressed as fold induction over tion (Figure 2). It should be noted that a normal controls (E). The data are expressed as the means Ϯ SEM (n ϭ 5 to 8). *P Ͻ 0.05 loss of WT1 does not necessarily indicate Ͻ Ͻ versus normal controls; **P 0.01 versus normal controls; †P 0.05 versus ADR alone. podocyte depletion, because podocyte ap- CTL, control; Pari., paricalcitol. optosis is an extremely rare event in this model.29 In that regard, the beneficial ef- ␤-catenin nuclear translocation in human proximal tubular fects of paricalcitol are likely mediated by its ability to preserve epithelial cells (HKC-8) as well (Figure 9F), and paricalcitol podocyte integrity, rather than by preventing podocyte loss. also induced VDR to physically interact with ␤-catenin after This view is further supported by the fact that delayed admin- ADR stimulation (Figure 9G). Altogether, it appears clear that istration of paricalcitol is able to reverse an established protein- paricalcitol, via VDR, exhibits dual effects on ␤-catenin signal- uria. Interestingly, this anti-proteinuric action of vitamin D ing by inhibiting Wnt expression and by sequestering ␤-cate- analogs is also reported in several clinical studies in patients nin transcriptional activity (Figure 9H). with chronic renal insufficiency,19,20,24,30 as well as in other animal models of proteinuric kidney diseases.31–35 Therefore, it is becoming clear that vitamin D analogs may represent a class DISCUSSION of anti-proteinuric agents that are quite effective in alleviating podocyte injury and proteinuria in different circumstances. Proteinuria, the clinical manifestation of defective glomerular The studies reported here likely offer significant, mechanis- filtration, is an early pathologic feature of many primary glo- tic insights into the mechanism by which vitamin D analogs merular diseases. It not only serves as a surrogate marker for protect podocytes from injury. We have recently shown that the progression and prognosis of kidney injury but also is an activation of the canonical pathway of Wnt/␤-catenin signal- important pathogenic mediator that triggers subsequent in- ing plays an imperative role in mediating podocyte dysfunc- flammatory and fibrotic responses in renal parenchyma. The tion.6 Modulation of this signal system in vivo by an array of results presented in this study demonstrate that paricalcitol, a genetic and pharmacologic maneuvers evidently influences the synthetic, low-calcemic vitamin D analog, possesses an im- development and severity of podocyte damage and protein- pressive renal protective efficacy in ADR nephropathy, a model uria.6 Notably, the importance of ␤-catenin in mediating characterized by initial podocyte injury, proteinuria, and late- podocyte injury is recently confirmed by an independent onset renal inflammation and fibrosis. The beneficial effects of study.36 Therefore, it is not surprising that targeting Wnt/␤- paricalcitol are likely mediated by its ability to inhibit Wnt catenin signaling by paricalcitol ameliorates proteinuria. In the expression and to block ␤-catenin–mediated gene transcrip- injured kidney, the expression of several Wnts is up-regulated

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Figure 6. Paricalcitol blocks renal ␤-catenin accumulation and activation after ADR injury. (A and B) Western blot analyses show renal ␤-catenin protein abundance at 5 weeks after ADR injection in different groups of mice as indicated. Whole-kidney lysates were immunoblotted with specific antibodies against ␤-catenin and GAPDH, respectively. Representative Western blots (A) and quantitative determination of ␤-catenin protein levels (B) are presented. The numbers (1, 2, and 3) denote each individual animal in a given group. The data are expressed as the means Ϯ SEM (n ϭ 5 to 8). **P Ͻ 0.01 versus normal controls; †P Ͻ 0.05 versus ADR alone. (C through F) Representative micrographs show ␤-catenin protein expression and localization in the kidneys at 5 weeks after ADR injection in different groups of mice. The arrows indicate glomeruli. (C) Normal controls. (D) ADR. (E) ADR plus paricalcitol. (F) Enlarged image of the boxed area in D. The arrowheads (yellow) indicate ␤-catenin–positive podocytes. Scale bar, 50 ␮m. CTL, control; Pari., paricalcitol.

(Figure 5). It should be pointed out that the expression pattern occurs in glomerular podocytes but also in tubular epithelial of specific Wnts in this study using whole-kidney lysates at 5 cells (Figure 9), as well as in colon carcinoma cells.14 Of note, weeks is quite different from that derived from the isolated previous studies show that calcitriol (1,25-dihydroxyvitamin 6 glomeruli at 1 day after ADR injection, consistent with the D3) inhibits Wnt signaling by inducing its antagonist DKK1 notion that Wnt expression is dynamic and changes with times gene expression in a human colon cancer cell line.37 However, during renal injury.7 Regardless of what specific Wnt is in- that mode of action is unlikely to be operative in the kidney, duced, however, ␤-catenin, the common downstream media- because paricalcitol does not induce the expression of any tor of the canonical Wnt signaling, is induced (Figures 6 and members of the DKK family in vivo (Figure 5). 8), indicating a robust activation of the canonical pathway of Of many Wnt/␤-catenin downstream target genes, Snail is Wnt signaling in this model. Interestingly, this Wnt/␤-catenin well characterized and mostly relevant to proteinuria and renal signaling is virtually blocked after paricalcitol treatment, un- fibrosis observed in ADR nephropathy.6,38 Recent studies sug- derscoring that the vitamin D analog is able to constrain the gest that podocytes also undergo EMT in response to injurious activity of Wnt/␤-catenin signaling in vivo. stimuli,39,40 in which Snail may play a role.41,42 Snail down- Our results indicate that paricalcitol not only prevents the regulates key epithelial markers such as E-cadherin by binding development of proteinuria after ADR injury but also induces to the E-box in the regulatory region of its target genes.43–45 We reversal of an established proteinuria (Figure 8). These find- have previously shown that ␤-catenin induces Snail expression ings are quite significant and obviously have clinical relevance. in glomerular podocytes,6 which in turn directly suppresses the It is conceivable that paricalcitol could inhibit Wnt/␤-catenin expression of nephrin.6,38,41 Overexpression of Snail is also suf- signaling at least by two different mechanisms (Figure 9H). On ficient to induce kidney injury and fibrosis, as illustrated in one hand, paricalcitol selectively suppresses the expression of Snail transgenic mice.46 Therefore, Snail could be a major multiple Wnt genes including Wnt4, Wnt7a, Wnt7b, and downstream effector of Wnt/␤-catenin signaling that mediates Wnt10a, whose expression is up-regulated after ADR injury. podocyte dysfunction and tubular EMT by virtue of its ability This action presumably prevents Wnt induction and ␤-catenin to repress nephrin and E-cadherin expression (Figure 9H). In- activation in the injured kidney after ADR injection in the first deed, renal Snail expression is markedly induced after ADR place. On the other hand, even after ␤-catenin is activated in an injection, and paricalcitol substantially suppresses its induc- established proteinuria, paricalcitol apparently has the ability tion. It is worthwhile to point out that Wnt signaling may to inhibit ␤-catenin–mediated gene transcription by inducing influence Snail protein abundance by both transcriptional and VDR binding to active, nuclear ␤-catenin (Figure 9). This leads post-translational mechanisms (Figure 7E), two distinct path- to the sequestration of the ␤-catenin transcriptional activity in ways regulated by ␤-catenin and glycogen synthase kinase-3␤ the nuclei. Such a mode of action of vitamin D analog not only (GSK-3␤), respectively. Although ␤-catenin directly induces

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nin/Snail signaling. Along this line, a recent study indicates that NF-␬B activation leads to Snail stabilization by preventing its degradation, which links inflammation to the major product of Wnt/␤-catenin signaling.50 Furthermore, Snail transcriptionally represses the expression of VDR,51 a potent inhibitor of Wnt/␤-catenin signaling. This creates a vicious cycle of vitamin D deficiency, Wnt/␤-catenin activation, and Snail induction in the state of chronic kidney diseases. Therefore, disruption of this cycle by vitamin D analog, as shown in this study, evidently silences Wnt/␤-catenin signaling and Figure 7. Paricalcitol suppresses renal Snail expression after ADR injury. (A and B) inhibits Snail expression, thereby preventing Western blot analyses show renal Snail protein expression at 5 weeks after ADR podocyte injury, proteinuria, and renal fibro- injection in different groups of mice as indicated. Representative Western blots (A) and sis in ADR nephropathy. quantitative determination of Snail protein levels (B) are presented. The numbers (1, 2, and 3) denote each individual animal in a given group. The data are expressed as the means Ϯ SEM (n ϭ 5 to 8). **P Ͻ 0.01 versus normal controls; ††P Ͻ 0.01 versus ADR CONCISE METHODS alone. (C and D) RT-PCR results show renal Snail mRNA expression in different groups of mice as indicated. Snail mRNA levels were determined by real-time RT-PCR and Animal Models ␤ expressed as fold induction over normal controls after normalization with -actin. The Mouse model of podocyte injury and protein- Ϯ ϭ Ͻ data are expressed as the means SEM (n 5 to 8). *P 0.05 versus normal controls; uria was established by intravenous injection of Ͻ †P 0.05 versus ADR alone. (E) Diagram shows the putative signaling pathways ADR, as described previously.6,25 Male BALB/c leading to Snail mRNA and protein induction in ADR nephropathy. CTL, control; Pari., mice weighing 20–22 g were obtained from paricalcitol. Harlan Sprague-Dawley (Indianapolis, Indi- Snail mRNA expression,6 Wnt-mediated GSK-3␤ inhibition ana). Three sets of animal experiments were performed. The first set results in Snail dephosphorylation, leading to its stabilization consisted of three groups of mice: (1) normal control (n ϭ 5); (2) by preventing ubiquitin-mediated degradation.47,48 Consistent ADR mice injected with vehicle (n ϭ 8); and (3) ADR mice injected with this notion, the magnitude of Snail protein induction is with paricalcitol (n ϭ 8). ADR (doxorubicin hydrochloride; Sigma, greater than that of its mRNA after ADR injury (Figure 7). St. Louis, Missouri) was administered by a single intravenous injec- The therapeutic efficacy of paricalcitol in ADR nephropathy tion at 10 mg/kg body wt. Paricalcitol (kindly provided by Abbott is impressive, which could involve multiple mechanisms. In Laboratories, Abbott Park, Illinois) was given by daily subcutaneous addition to modulating Wnt/␤-catenin signaling, we cannot injection at 50 ng/kg body wt, starting at the time when ADR was exclude the possibility that paricalcitol may elicit its beneficial administered. The dose of paricalcitol was chosen on the basis of our activities by other routes as well. In that regard, paricalcitol has pilot experiments. At 5 weeks after ADR injection, all of the mice were been shown to inhibit renal inflammation by promoting VDR- sacrificed. The second set of experiments consisted of the same three mediated sequestration of NF-␬B signaling,49 consistent with a groups as the first set, but the animals (n ϭ 6) were sacrificed at 7 days reduced renal infiltration of macrophages and decreased ex- after ADR injection. The third set of experiments consisted of five groups pression of proinflammatory cytokines in this study. Further- in which paricalcitol was administered either 1 day before or 2 and 6 days more, paricalcitol is able to attenuate renal interstitial fibrosis after ADR injection, respectively, and the animals were sacrificed at 3 by blocking tubular EMT,43,45 a process in which ␤-catenin and weeks after ADR injection. The details of the experimental design for this Snail play a critical role. Because renal inflammation and fibro- set of experiments are presented in Figure 8A. Urine and kidney tissue sis are late-onset events, secondary to podocyte injury and pro- were collected for various analyses. All of the animal studies were per- teinuria in this model, it is plausible that paricalcitol inhibition formed by use of the procedures approved by the Institutional Animal of Wnt/␤-catenin signaling may play a primary and predomi- Care and Use Committee at the University of Pittsburgh. nant role in protecting the kidney from developing ADR nephropathy. However, paricalcitol seems to inhibit ␤-catenin Urinary Albumin and Creatinine Assay signaling after ADR injury in tubular epithelial cells as well Urine albumin was measured by using a mouse albumin ELISA quanti- (Figure 9), suggesting that some of its effects on the tubuloint- tation kit, according to the manufacturer’s protocol (Bethyl Laboratories, erstitium may be direct events. Inc., Montgomery, Texas). Urine creatinine was determined by a routine We should point out that the signals controlling podocyte procedure as described previously.52 Urinary proteins were also analyzed dysfunction, inflammation, and fibrosis may cross-talk with by SDS-PAGE after normalization to urinary creatinine. After separation each other and are likely integrated in the path of Wnt/␤-cate- by SDS-PAGE, urine proteins were stained with Coomassie Blue R-250.

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Figure 8. Paricalcitol induces reversal of an established proteinuria in ADR nephropathy. (A) Diagram shows the experimental design. The arrows indicate the starting point of daily injections of paricalcitol, whereas heavy arrowheads denote the single injection of ADR. (B) Urinary albumin levels in different groups as indicated. Mouse urine was collected weekly after ADR injection, and urinary albumin level was expressed as mg/mg creatinine. *P Ͻ 0.05 group 3 versus group 2; †P Ͻ 0.05 group 4 versus group 2; #P Ͻ 0.05 group 5 versus group 2 (n ϭ 5 to 6). (C) SDS-PAGE shows the abundance and composition of urinary proteins in different groups of mice at 21 days after ADR injection. Urine samples after normalization to creatinine were analyzed on SDS-PAGE, with BSA (1 ␮g) loaded on the adjacent lane. The numbers (1 and 2) indicate each individual, representative animal in a given group. (D) Representative micrographs demonstrate kidney histology in different groups of mice. Kidney sections were stained with periodic acid-Schiff reagent. (Panel a) Control. (Panel b) ADR alone. (Panel c) ADR plus paricalcitol at Ϫ1 day. (Panel d) ADR plus paricalcitol at ϩ 2 days. (Panel e) ADR plus paricalcitol at ϩ 6 days. Scale bar, 100 ␮m. (E and F) Western blot analyses show renal ␤-catenin protein abundance at 3 weeks after ADR injection in different groups of mice as indicated. Whole-kidney lysates were immunoblotted with specific antibodies against ␤-catenin and GAPDH, respectively. Representative Western blots (E) and quantitative determination of ␤-catenin protein levels (F) are presented. The numbers (1, 2, and 3) denote each individual animal in a given group. The data are expressed as the means Ϯ SEM (n ϭ 5 to 6). *P Ͻ 0.05 versus ADR alone. CTL, control; Veh., vehicle; Pari., paricalcitol.

Histology and Immunohistochemical Staining 30 minutes, the slides were immunostained with primary antibodies Paraffin-embedded mouse kidney sections (3-␮m thickness) were pre- against nephrin (catalog number 20R-NP002; Fitzgerald Industries pared by a routine procedure. The sections were stained with hematoxy- International, Inc., Concord, Massachusetts), podocin (catalog num- lin-eosin, periodic acid-Schiff reagent by standard protocol. Kidney sec- ber SC-22298), and WT1 (catalog number SC-192; Santa Cruz Bio- tions were also subjected to Masson-trichrome staining for assessing technology, Santa Cruz, California). The slides were viewed under a collagen deposition and fibrotic lesions. Quantitation of the fibrotic area Leica TCS-SL confocal microscope. was carried out by a computer-aided morphometric analysis (Meta- Morph; Universal Imaging Co., Downingtown, Pennsylvania), as de- Western Blot Analysis scribed previously.43 Immunohistochemical staining was performed using Glomeruli were isolated by differential serving technique according to a routine protocol.43 The antibodies used were as follows: affinity-purified the method described elsewhere.53 The isolated glomeruli were lysed anti-mouse F4/80 antigen (catalog number 14-4801; eBioscience, San Diego, with radioimmune precipitation assay buffer containing 1% NP40, California) and rabbit polyclonal anti-␤-catenin antibody (ab15180; Abcam, 0.1% SDS, 100 ␮g/ml PMSF, 1% protease inhibitor cocktail, and 1% Cambridge, Massachusetts). phosphatase I and II inhibitor cocktail (Sigma) in PBS on ice. The supernatants were collected after centrifugation at 13,000 ϫ g at 4°C Immunofluorescence Staining and Confocal Microscopy for 20 minutes. Whole-kidney lysates were prepared using the same Kidney cryosections were fixed with 3.7% paraformalin for 15 min- procedures. Cultured mouse podocytes were lysed in SDS sample utes at room temperature. After blocking with 10% donkey serum for buffer. Protein expression was analyzed by Western blot analysis as

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Figure 9. Paricalcitol induces VDR to interact with ␤-catenin and sequestrate its transcription activity. (A and B) Western blot analyses show the nuclear and total cellular ␤-catenin and VDR abundances after various treatments as indicated. Mouse podocytes were Ϫ7 pretreated without or with paricalcitol (Pari., 10 M) for 1 hour and then incubated with ADR (10 ␮g/ml) for an additional 1 hour. Nuclear protein preparation (A) and whole cell lysates (WCL) (B) were immunoblotted (IB) with antibodies against ␤-catenin and VDR, respectively. TBP and GAPDH were used for normalization of nuclear protein and WCL, respectively. n, nuclear; T, total. (C) Coimmunoprecipitation (IP) reveals that paricalcitol induced VDR/␤-catenin complex formation in podocytes. Cell lysates were prepared after various treatments as indicated and immunoprecipitated with specific antibody against VDR, followed by immunoblotting for ␤-catenin. Cellular ␤-catenin levels after various treatments were assessed by routine Western blot analysis of WCLs. (D) Graphic presentation shows the relative abundance of VDR/␤-catenin complex after various treatments as indicated. The data are expressed as the means Ϯ SEM (n ϭ 3). *P Ͻ 0.05 versus controls. (E) Paricalcitol inhibits ␤-catenin–mediated gene transcription. Podocytes were transfected with TOP-flash reporter plasmid in the absence or presence of stabilized ␤-catenin expression vector (pDel-␤-cat). Twenty-four Ϫ7 hours after transfection, the cells were incubated with or without paricalcitol (10 M). Relative luciferase was reported as the means Ϯ SEM (n ϭ 3). **P Ͻ 0.01 versus controls; ††P Ͻ 0.01 versus pDel-␤-cat alone. (F) ADR also induces ␤-catenin nuclear translocation in proximal tubular epithelial cells (HKC-8). HKC-8 cells were treated with ADR (10 ␮g/ml) for different periods of time as indicated. Nuclear ␤-catenin levels were assessed by immunoblotting of nuclear lysates with antibodies against ␤-catenin and TBP, respectively. (G) Paricalcitol also induces VDR/␤-catenin complex formation in proximal tubular epithelial cells (HKC-8). The cells were pretreated with paricalcitol for 1 hour, followed by incubation with ADR (10 ␮g/ml) for 1 hour. (H) Schematic diaphragm shows that paricalcitol, via VDR, inhibits Wnt expression and blocks ␤-catenin–mediated gene transcription. described previously.7 The primary antibodies used were as follows: Wisconsin). Real-time RT-PCR was performed on ABI PRISM 7000 anti-nephrin (Fitzgerald Industries International), anti-podocin, an- sequence detection system (Applied Biosystems, Foster City, Califor- ti-WT1, anti-VDR (SC-1008), and anti-actin (SC-1616) (Santa Cruz nia) as described previously.54 The PCR mixture in a 25-␮l volume Biotechnology), anti-␤-catenin (catalog number 610154; BD Trans- contained 12.5 ␮lof2ϫ SYBR Green PCR Master Mix (Applied Bio- duction Laboratories, San Jose, California), anti-␣-SMA (clone 1A4; systems), 5 ␮l of diluted RT product (1:10), and 0.5 ␮M sense and Sigma), anti-Snail (ab17732; Abcam), anti-TATA-binding protein antisense primer sets. The sequences of the primer pairs used in real- (TBP) (catalog number ab181–100; Abcam), and anti-glyceralde- time PCR were given in Supplemental Table 1. PCR was run by using hyde-3-phosphate dehydrogenase (GAPDH) (Ambion, Austin, standard conditions. After sequential incubations at 50°C for 2 min- Texas). utes and 95°C for 10 minutes, respectively, the amplification protocol consisted of 50 cycles of denaturing at 95°C for 15 seconds and Real-Time RT-PCR annealing and extension at 60°C for 60 seconds. The standard Total RNA isolation and real-time RT-PCR were carried out by the curve was made from series dilutions of template cDNA. The procedures described previously.54 Briefly, the first strand cDNA syn- mRNA levels of various genes were calculated after normalizing thesis was carried out by using a reverse transcription system kit ac- with ␤-actin. Expression of Wnt mRNA levels was determined as cording to the instructions of the manufacturer (Promega, Madison, described previously.7

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Ϫ7 Cell Culture and Treatment paricalcitol (10 M) as indicated. Luciferase assay was performed us- The conditionally immortalized mouse podocyte cell line was kindly ing a dual luciferase assay system kit according to the manufacturer’s provided by Dr. Peter Mundel (University of Miami, Miami, Florida), protocols (Promega). Relative luciferase activity (arbitrary units) as described previously.41,55 The cells were cultured at 33°C in RPMI was reported as fold induction over the controls after normalizing 1640 medium supplemented with 10% fetal bovine serum and recom- for transfection efficiency. binant IFN-␥ (Invitrogen, Carlsbad, California). To induce differentiation, podocytes were grown under nonpermissive conditions at Statistical Analyses 37°C in the absence of IFN-␥. After serum starvation for 16 hours, the Statistical analyses of the data were carried out using SigmaStat soft- Ϫ7 cells were treated with paricalcitol (10 M). Human proximal tubular ware (Jandel Scientific, San Rafael, California). Comparison between epithelial cells (HKC, clone-8) were provided by Dr. L. Racusen groups was made using one-way ANOVA followed by a Student- (Johns Hopkins University, Baltimore, Maryland). Cell culture was Newman-Kuel’s test. P Ͻ 0.05 was considered significant. carried out according to the procedures described previously43 and treated with paricalcitol. Whole-cell lysates were prepared and subjected to coimmunoprecipitation and Western blot analyses. ACKNOWLEDGMENTS

Nuclear Protein Preparation This work was supported by National Institutes of Health Grants Nuclear protein preparation was carried out according to the proce- DK061408, DK064005, and DK071040 and a Grant-in-Aid from the dure described previously.56 Briefly, mouse podocytes or HKC-8 cells Abbott Laboratories. C.D. was supported by American Heart Associ- after various treatments as indicated were washed twice with cold PBS ation Beginning Grant-in-Aid 0865392D and the University of Pitts- and scraped off the plate with a rubber policeman. After centrifuga- burgh Medical Center Health System Competitive Medical Research tion, the cell pellets were resuspended in Buffer A (10 mM HEPES, pH Fund. 7.9, 1.5 mM MgCl2,10mM KCl, 0.5% NP-40, and 1% protease inhibitor cocktail [Sigma]) and lysed with homogenizer. The cell nuclei were collected by centrifugation at 5000 rpm for 15 minutes and DISCLOSURES washed with Buffer B (10 mM HEPES, pH 7.9, 1.5 mM MgCl2,10mM KCl, and 1% protease inhibitor cocktail). The nuclei were lysed in SDS None. sample buffer. For loading control of nuclear protein, the blots were stripped and reprobed with antibody against the TBP. REFERENCES Coimmunoprecipitation Immunoprecipitation was carried out by using an established method.52 1. Shankland SJ: The podocyte’s response to injury: Role in proteinuria Briefly, mouse podocytes and HKC-8 cells after various treatments and glomerulosclerosis. Kidney Int 69: 2131–2147, 2006 2. Wiggins RC: The spectrum of podocytopathies: A unifying view of were lysed on ice in 1 ml of nondenaturing lysis buffer that contained glomerular diseases. Kidney Int 71: 1205–1214, 2007 1% Triton X-100, 0.01 M Tris-HCl (pH 8.0), 0.14 M NaCl, 0.025% 3. Patrakka J, Tryggvason K: New insights into the role of podocytes in NaN3, 1% protease inhibitors cocktail, and 1% phosphatase inhibi- proteinuria. Nat Rev Nephrol 5: 463–468, 2009 tors cocktail I and II (Sigma). After preclearing with normal IgG, cell 4. Abbate M, Zoja C, Remuzzi G: How does proteinuria cause progres- lysates (0.5 mg of protein) were incubated overnight at 4°C with 4 ␮g sive renal damage? J Am Soc Nephrol 17: 2974–2984, 2006 5. Zandi-Nejad K, Eddy AA, Glassock RJ, Brenner BM: Why is proteinuria of anti-VDR (Santa Cruz Biotechnology), followed by precipitation ␮ an ominous biomarker of progressive kidney disease? Kidney Int with 30 l of protein A/G Plus-agarose for1hat4°C. The precipitated Suppl S76–S89, 2004 complexes were separated on SDS-PAGE and immunoblotted with 6. Dai C, Stolz DB, Kiss LP, Monga SP, Holzman LB, Liu Y: Wnt/␤-catenin anti-␤-catenin antibody. signaling promotes podocyte dysfunction and albuminuria. J Am Soc Nephrol 20: 1997–2008, 2009 7. He W, Dai C, Li Y, Zeng G, Monga SP, Liu Y: Wnt/␤-catenin signaling Transfection and Luciferase Assay promotes renal interstitial fibrosis. J Am Soc Nephrol 20: 765–776, ␤ The effect of paricalcitol on -catenin–mediated gene transcription 2009 was assessed by using the TOP-flash TCF reporter plasmid containing 8. Schmidt-Ott KM, Barasch J: WNT/beta-catenin signaling in nephron two sets of three copies of the TCF binding site upstream of the thy- progenitors and their epithelial progeny. Kidney Int 74: 1004–1008, midine kinase (TK) minimal promoter and luciferase open reading 2008 9. Moon RT, Kohn AD, De Ferrari GV, Kaykas A: WNT and beta-catenin frame (Millipore, Billerica, Massachusetts). Podocytes were cotrans- signalling: Diseases and therapies. Nat Rev Genet 5: 691–701, 2004 fected by using Lipofectamine 2000 reagent (Invitrogen) with TOP- 10. Surendran K, Schiavi S, Hruska KA: Wnt-dependent beta-catenin sig- flash plasmid (1 ␮g) and VDR expression vector (1 ␮g) in the absence naling is activated after unilateral ureteral obstruction, and recombi- or presence of the stabilized ␤-catenin expression vector (pDel-␤- nant secreted frizzled-related protein 4 alters the progression of renal cat). An internal control reporter plasmid (0.1 ␮g) Renilla reniformis fibrosis. J Am Soc Nephrol 16: 2373–2384, 2005 11. Surendran K, McCaul SP, Simon TC: A role for Wnt-4 in renal fibrosis. luciferase driven under TK promoter (pRL-TK; Promega) was also Am J Physiol Renal Physiol 282: F431–F441, 2002 cotransfected for normalizing the transfection efficiency. The trans- 12. von Toerne C, Schmidt C, Adams J, Kiss E, Bedke J, Porubsky S, Gretz fected cells were incubated in serum-free medium without or with N, Lindenmeyer MT, Cohen CD, Grone HJ, Nelson PJ: Wnt pathway

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J Am Soc Nephrol 22: 90–103, 2011 Paricalcitol Blocks Wnt/␤-Catenin Signaling 103