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J Am Soc Nephrol. Jan 2011; 22(1): 90–103.
PMCID: PMC3014038

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

Abstract

Recent studies implicate Wnt/β-catenin signaling in podocyte dysfunction. Because vitamin D analogs can inhibit β-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-β1, 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 β-catenin, predominantly in podocytes and tubular epithelial cells, accompanied renal injury; paricalcitol largely abolished this induction of renal β-catenin and inhibited renal expression of Snail, a downstream effector of Wnt/β-catenin signaling. Administration of paricalcitol also ameliorated established proteinuria. In vitro, paricalcitol induced a physical interaction between the vitamin D receptor and β-catenin in podocytes, which led to suppression of β-catenin–mediated gene transcription. In summary, these findings suggest that paricalcitol prevents podocyte dysfunction, proteinuria, and kidney injury in adriamycin nephropathy by inhibiting Wnt/β-catenin signaling.

Proteinuria is an early and predominant pathologic feature of a wide variety of primary glomerular diseases that progress to end-stage renal failure. Increasing evidence suggests that podocyte injury is one of the major causes leading to defective glomerular filtration, which results in proteinuria.13 It has been well documented that proteinuria not only is a marker for the progression of chronic kidney diseases (CKD) but also acts as a pathogenic mediator that incites renal inflammation and promotes tubular injury and interstitial fibrosis.4,5 Despite the fact that the importance of podocyte injury in proteinuria is well recognized, the mechanisms and signal pathways leading to podocyte damage in the vast majority of proteinuric kidney disorders remain poorly understood. We have recently shown that Wnt/β-catenin signaling plays a critical role in promoting podocyte injury, proteinuria, and renal fibrosis.6,7 In this context, it is conceivable that developing a strategy aimed to target the Wnt/β-catenin signal pathway may be a plausible approach for the treatment of proteinuric kidney disorders.

Wnt/β-catenin is an evolutionarily conserved cellular signaling system that plays an essential role in diverse array of biologic processes such as organogenesis, tissue homeostasis, and pathogenesis of many human diseases.8,9 Aberrant regulation of Wnt/β-catenin has been implicated in many types of kidney diseases including obstructive nephropathy, chronic allograft nephropathy, diabetic nephropathy, polycystic kidney disease, focal and segmental glomerulosclerosis, and adriamycin nephropathy.6,7,1012 Wnt proteins transmit their signal across the plasma membrane through interacting with the Frizzled (Fzd) receptors, as well as their coreceptors, members of the LDL receptor-related protein 5/6. Upon binding to their receptors/coreceptors, Wnt proteins induce a series of downstream signaling events, resulting in β-catenin dephosphorylation and stabilization. This allows β-catenin to translocate into the nuclei, wherein it binds to T cell factor (TCF)/lymphoid enhancer-binding factor to stimulate the transcription of Wnt target genes.13 On the basis of this canonical pathway of Wnt signaling, it is conceivable that either inhibiting Wnt expression or repressing β-catenin transcriptional activity could be an effective way to control the Wnt/β-catenin signaling.

Earlier studies indicate that vitamin D analogs are able to promote the differentiation of colon carcinoma cells by inhibiting β-catenin signaling.14 This action of vitamin D appears to be mediated by ligand-activated vitamin D receptor (VDR) competing with transcription factor TCF-4 for β-catenin binding. These observations suggest that vitamin D and its receptor compose an endogenous negative regulator that tightly controls β-catenin signaling.15,16 Interestingly, deficiency in vitamin D and its active metabolites is highly prevalent in advanced stage CKD,17,18 in which Wnt/β-catenin signaling is activated.7,11,12 Consistently, administration of vitamin D analogs are able to reduce proteinuria and promote overall survival in patients with CKD by a mechanism that is independent of serum parathyroid hormone, phosphorus, and calcium levels.1924 Taken together, these results led us to hypothesize that administration of vitamin D analog might be able to effectively prevent podocyte dysfunction, proteinuria, and kidney injury by modulating Wnt/β-catenin signaling.

Here we examined the therapeutic effects of paricalcitol (19-nor-1,25-hydroxy-vitamin D2), a synthetic and active vitamin D analog, in adriamycin (ADR) nephropathy. Our data demonstrate that paricalcitol mitigates proteinuria and kidney injury by inhibiting Wnt/β-catenin signaling. These studies indicate that blocking Wnt/β-catenin signaling is a plausible strategy for therapeutic intervention of proteinuric kidney disorders.

RESULTS

Paricalcitol Ameliorates Proteinuria and Kidney Injury in Adriamycin Nephropathy

We investigated the effects of paricalcitol on ADR nephropathy, a model characterized by initial podocyte injury and albuminuria and subsequent renal inflammation and fibrosis.25,26 Of interest, three of eight mice with severe proteinuria in ADR group died between 3 and 5 weeks after ADR injection, whereas all eight mice survived in the ADR group receiving paricalcitol. As shown in Figure 1A, urinary albumin levels markedly elevated at 5 weeks after ADR injection, and administration of paricalcitol largely prevented proteinuria in this model. Kidney histology by Masson-trichrome staining revealed clearly visible nephropathy at 5 weeks after ADR injection, characterized by the fibrotic lesions in the glomeruli (Figure 1B, arrow), tubular dilation with proteinous fluid in the lumens (Figure 1B, asterisks), as well as expanded interstitial space. Consistent with the proteinuria data, administration of paricalcitol ameliorated kidney injury after ADR injection (Figure 1B). Quantitative determination of kidney fibrotic lesion among different groups at 5 weeks after ADR injection is presented in Figure 1C. To assess more acute effects of paricalcitol on the development of proteinuria, another set, short duration of animal experiments was performed. As shown in Figure 1D, robust albuminuria was evident in mice at 7 days after ADR injection, and paricalcitol also significantly reduced urinary albumin level in this setting.

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 ...

Paricalcitol Prevents Podocyte Injury and Reduces Glomerular Lesions In Vivo

Because podocyte injury is an early and predominant pathologic feature of this model,6,25 we next investigated the effects of paricalcitol on podocyte damage and glomerular lesions in vivo. As shown in Figure 2A, comparing with normal controls, podocyte slit diaphragm–associated proteins nephrin and podocin were substantially down-regulated in the kidney at 5 weeks after ADR injection, as illustrated by immunofluorescence staining. Western blot analyses of the isolated glomeruli from different groups of mice produced similar results (Figure 2B). However, these slit diaphragm–associated proteins were restored after paricalcitol treatment (Figure 2, A and B), indicating an effective preservation of podocyte integrity.

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 ...

We also examined the expression of Wilms tumor 1 (WT1) protein, a pivotal transcription factor that is essential for the maintenance of the differentiated features of adult podocytes.27,28 As illustrated in Figure 2 (A and B), WT1 protein expression was also markedly suppressed in the glomeruli after ADR injury, and paricalcitol treatment restored WT1 protein expression. Of note, despite a significant decrease in the numbers of the WT1-positive cells, podocyte apoptosis as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining was extremely rare (<2 per 100 glomerular cross-sections) at 5 weeks after ADR injection (data not shown).

Figure 2 (C through E) shows that podocyte injury and glomerular lesions were an early event in this model. At 7 days after ADR injection, nephrin, podocin, and WT1 were already down-regulated, and paricalcitol was able to largely preserve their expression (Figure 2, C through E).

Paricalcitol Inhibits Renal Inflammation

We next examined the effects of paricalcitol on renal inflammation at 5 weeks after ADR injection, because an increased renal infiltration of inflammatory cells is a pathologic feature of this model. To this end, we initially investigated the expression of several proinflammatory cytokines including the regulated on activation normal T cell expressed and secreted (RANTES), also known as CC-chemokine ligand 5, TNF-α, and monocyte chemotactic protein-1 (MCP-1), also known as CC-chemokine ligand 2. As shown in Figure 3A, at 5 weeks after ADR injection, the renal mRNA levels for RANTES, TNF-α, and MCP-1 were markedly up-regulated. Administration of paricalcitol substantially inhibited renal expression of these proinflammatory cytokines (Figure 3, B and C), as determined by quantitative real-time reverse transcriptase (RT)-PCR approach. Consistently, immunohistochemical staining for F4/80 antigen, a marker for myeloid cells including monocytes/macrophages and dendritic cells, showed that an increased renal infiltration of the F4/80-positive cells in kidney parenchyma after ADR injection (Figure 3, D and E). Notably, virtually all F4/80-positive cells were found in the interstitium but not in the glomeruli (Figure 3E). Paricalcitol effectively blocked renal infiltration of these F4/80-positive inflammatory cells (Figure 3F).

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-α, and MCP-1 at 5 weeks after ADR injection in different ...

Paricalcitol Reduces Renal Fibrotic Lesions after Adriamycin Injury

Because ADR injury inevitably leads to renal fibrotic lesions, we next examined the effects of paricalcitol on renal fibrosis in this model. To this end, we initially investigated the expression of TGF-β1 and connective tissue growth factor (CTGF), two major fibrogenic cytokines that are involved in the pathogenesis of a wide array of CKD. As shown in Figure 4 (A through C), real-time RT-PCR analyses demonstrated that both TGF-β1 and CTGF mRNA levels were increased in the kidney at 5 weeks after ADR injection, and paricalcitol significantly abrogated their induction. Analyses of the expression of several interstitial matrix genes such as fibronectin and type I and type III collagen in different groups of mice also indicated that paricalcitol was able to inhibit the mRNA expression of major interstitial matrix genes induced by ADR (Figure 4, D through G). These results are consistent with and supported by an altered renal collagen deposition revealed by Masson-trichrome staining (Figure 1, B and C).

Figure 4.
Paricalcitol inhibits renal expression of TGF-β1, 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 ...

We further examined renal expression of α-smooth muscle actin (α-SMA) and myofibroblast activation in different groups of mice. As shown in Figure 4 (H and I), renal α-SMA protein levels were dramatically increased after ADR injury, suggesting myofibroblast activation in this model. This induction of renal α-SMA, however, was largely abolished by paricalcitol (Figure 4, H and I). Similar results were obtained when examining myofibroblast activation by immunofluorescence staining for α-SMA protein (data not shown).

Paricalcitol Represses Renal Expression of Wnt Genes

To provide mechanistic insights into the renal protective efficacy of paricalcitol in ADR nephropathy, we investigated its effects on the activation of Wnt/β-catenin signaling, because recent studies suggest a critical role of this signal pathway in podocyte dysfunction and renal fibrosis.6,7 A comprehensive analysis of all 19 Wnt genes has demonstrated that numerous Wnts were up-regulated in the kidney in this model, as reported previously.7 We found that paricalcitol could specifically inhibit renal expression of multiple Wnts, including Wnt4, Wnt7a, Wnt7b, and Wnt10a (Figure 5, A through C). However, paricalcitol appeared not to suppress Wnt3 expression; rather, it slightly induced its expression in this model (Figure 5, A and C). These results suggest that paricalcitol is able to selectively inhibit specific Wnt expression induced after ADR injury.

Figure 5.
The expression of Wnt genes is selectively inhibited by paricalcitol in the kidney after ADR injury. (A) Representative RT-PCR results show renal mRNA expression of various Wnt genes in different groups of mice as indicated at 5 weeks after ADR injection. ...

We also examined the expression of the members of the Dickkopf (DKK) family of endogenous Wnt antagonists in different groups of mice. As shown in Figure 5 (D and E), although ADR injury also caused an induction of these DKK genes, paricalcitol did not significantly affect their expression after ADR administration.

Paricalcitol Blocks β-Catenin Activation and Suppresses Its Downstream Snail Expression

Because β-catenin is the principal mediator of the canonical Wnt signaling, we next examined its regulation in ADR nephropathy. As shown in Figure 6 (A and B), Western blot analyses revealed a dramatic increase in renal β-catenin protein abundance at 5 weeks after ADR injection. Quantitative determination showed a more than 150-fold induction of β-catenin protein over the controls in this model (Figure 6B). Immunohistochemical staining demonstrated that β-catenin was predominantly localized at renal tubular epithelial cells and glomerular podocytes (Figure 6, D and F), whereas the staining for β-catenin in normal kidney was weak (Figure 6C). Cytoplasmic and nuclear staining of β-catenin was clearly visible in glomerular podocytes (Figure 6F, yellow arrowheads), as well as in renal tubular epithelia, suggesting the activation of this signaling in these cells. Of interest, administration of paricalcitol largely prevented β-catenin induction and activation (Figure 6, A, B and E).

Figure 6.
Paricalcitol blocks renal β-catenin accumulation and activation after ADR injury. (A and B) Western blot analyses show renal β-catenin protein abundance at 5 weeks after ADR injection in different groups of mice as indicated. Whole-kidney ...

We further examined the expression of Snail, a downstream mediator of the Wnt/β-catenin signaling in podocytes.6 As shown in Figure 7 (A and B), Snail protein was markedly induced in the injured kidney after ADR injection; and administration of paricalcitol largely blocked renal Snail induction. Similarly, renal Snail mRNA was also induced, to a lesser extent, after ADR injury, which was blocked by paricalcitol (Figure 7, C and D). Figure 7E shows the putative signaling pathways leading to Snail induction by ADR. These results suggest that paricalcitol is able to target a key pathogenic signaling by inhibiting Wnt/β-catenin and its downstream Snail.

Figure 7.
Paricalcitol suppresses renal Snail expression after ADR injury. (A and B) Western blot analyses show renal Snail protein expression at 5 weeks after ADR injection in different groups of mice as indicated. Representative Western blots (A) and quantitative ...

Paricalcitol Reverses an Established Proteinuria

We next investigated whether delayed administration of paricalcitol is still effective in ameliorating proteinuria, a scenario that is obviously of clinical relevance. As depicted in Figure 8A, several different treatment protocols were used. In the preventive protocol (group 3), paricalcitol was commenced 1 day before ADR injection. The mice in groups 4 and 5 were given paricalcitol starting at either 2 days after ADR, a time point when albuminuria is just about to emerge, or 6 days after ADR, a time point when robust albuminuria is already established in this model,6,29 respectively. As shown in Figure 8B, albuminuria was significantly reduced at 7 days after ADR when the preventive protocol was used (group 3). Interestingly, urinary albumin levels also started to decline, albeit not statistically significantly (P = 0.155, n = 6), in just 1 day after paricalcitol administration starting at 6 days after ADR (group 5) compared with ADR alone (group 2). At 14 and 21 days after ADR, albuminuria was significantly reduced in all three groups that received paricalcitol, regardless of the starting time points when paricalcitol administration was initiated (Figure 8B). In fact, the levels of urinary albumin in these paricalcitol-treated groups were indistinguishable. Notably, a time-dependent regression of proteinuria was observed in all three groups that received paricalcitol (Figure 8B). Analyses of urinary proteins by SDS-PAGE revealed the similar results (Figure 8C), suggesting that paricalcitol is able to ameliorate and reverse an established proteinuria. Consistently, renal histology showed that significant morphologic lesions were evident in the kidney at 3 weeks after ADR injection (Figure 8D, panel b), and these histologic injuries were markedly mitigated in all three groups that received paricalcitol (Figure 8D, panels c through e).

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 ...

We further examined renal β-catenin expression in different groups. As shown in Figure 8 (E and F), β-catenin abundance in the injured kidney at 3 weeks after ADR was increased approximately 45-fold over the controls. Treatment with paricalcitol starting at 1 day before or 2 days after ADR, respectively, significantly prevented renal β-catenin induction. However, delayed administration of paricalcitol at 6 days after ADR was clearly less effective in inhibiting β-catenin expression (Figure 8, E and F). This discrepancy between renal β-catenin abundance (Figure 8, E and F) and the severity of albuminuria and histologic lesions (Figure 8, B through D) in three paricalcitol-treated groups raise the possibility that paricalcitol may also directly disrupt β-catenin signaling, in addition to inhibiting Wnt expression and β-catenin accumulation.

Paricalcitol Induces VDR to Interact with β-Catenin and Sequestrate Its Transcription Activity

To explore whether paricalcitol can directly affect β-catenin–mediated signaling, we used in vitro cultured mouse podocytes as a model system. We first examined whether paricalcitol blocked β-catenin nuclear translocation, an obligatory step for β-catenin to control its target gene transcription in the nucleus. As shown in Figure 9A, treatment of mouse podocytes with ADR induced β-catenin activation and its nuclear translocation, as nuclear β-catenin level was induced. Similarly, incubation of podocytes with paricalcitol induced VDR nuclear translocation (Figure 9A). However, it appeared that pretreatment with paricalcitol did not block β-catenin nuclear translocation triggered by ADR in podocytes (Figure 9A). Of note, neither paricalcitol nor ADR affect total cellular levels of VDR and β-catenin after a short period of incubation (Figure 9B). Given that both VDR and β-catenin undergo nuclear translocation after stimulation, we next tested whether activated VDR interacts with nuclear β-catenin by using a coimmunoprecipitation approach. As shown in Figure 9 (C and D), incubation of mouse podocytes with paricalcitol induced VDR to interact with β-catenin, as shown by increased VDR/β-catenin complex formation after paricalcitol stimulation. We further assessed the functional consequence of VDR/β-catenin interaction by examining the β-catenin–mediated gene transcription in a luciferase reporter system. As shown in Figure 8E, paricalcitol could significantly repress β-catenin–mediated gene transcription in cultured podocytes. Similarly, ADR induced β-catenin nuclear translocation in human proximal tubular epithelial cells (HKC-8) as well (Figure 9F), and paricalcitol also induced VDR to physically interact with β-catenin after ADR stimulation (Figure 9G). Altogether, it appears clear that paricalcitol, via VDR, exhibits dual effects on β-catenin signaling by inhibiting Wnt expression and by sequestering β-catenin transcriptional activity (Figure 9H).

Figure 9.
Paricalcitol induces VDR to interact with β-catenin and sequestrate its transcription activity. (A and B) Western blot analyses show the nuclear and total cellular β-catenin and VDR abundances after various treatments as indicated. Mouse ...

DISCUSSION

Proteinuria, the clinical manifestation of defective glomerular filtration, is an early pathologic feature of many primary glomerular diseases. It not only serves as a surrogate marker for the progression and prognosis of kidney injury but also is an important pathogenic mediator that triggers subsequent inflammatory and fibrotic responses in renal parenchyma. The results presented in this study demonstrate that paricalcitol, a synthetic, low-calcemic vitamin D analog, possesses an impressive renal protective efficacy in ADR nephropathy, a model characterized by initial podocyte injury, proteinuria, and late-onset renal inflammation and fibrosis. The beneficial effects of paricalcitol are likely mediated by its ability to inhibit Wnt expression and to block β-catenin–mediated gene transcription (Figure 9H). These studies underscore that vitamin D is a potent endogenous, natural antagonist of Wnt/β-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. Albuminuria, 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 conceivable that the reno-protective effect of paricalcitol may be primarily attributable to its prevention of podocyte injury. This notion is further substantiated by the observations that paricalcitol prevents the loss of podocyte-specific nephrin, podocin, and WT1 as early as 7 days after ADR injection (Figure 2). It should be noted that a loss of WT1 does not necessarily indicate podocyte depletion, because podocyte apoptosis is an extremely rare event in this model.29 In that regard, the beneficial effects of paricalcitol are likely mediated by its ability to preserve podocyte integrity, rather than by preventing podocyte loss. This view is further supported by the fact that delayed administration of paricalcitol is able to reverse an established proteinuria. Interestingly, this anti-proteinuric action of vitamin D analogs is also reported in several clinical studies in patients with chronic renal insufficiency,19,20,24,30 as well as in other animal models of proteinuric kidney diseases.3135 Therefore, it is becoming clear that vitamin D analogs may represent a class of anti-proteinuric agents that are quite effective in alleviating podocyte injury and proteinuria in different circumstances.

The studies reported here likely offer significant, mechanistic insights into the mechanism by which vitamin D analogs protect podocytes from injury. We have recently shown that activation of the canonical pathway of Wnt/β-catenin signaling plays an imperative role in mediating podocyte dysfunction.6 Modulation of this signal system in vivo by an array of genetic and pharmacologic maneuvers evidently influences the development and severity of podocyte damage and proteinuria.6 Notably, the importance of β-catenin in mediating podocyte injury is recently confirmed by an independent study.36 Therefore, it is not surprising that targeting Wnt/β-catenin signaling by paricalcitol ameliorates proteinuria. In the injured kidney, the expression of several Wnts is up-regulated (Figure 5). It should be pointed out that the expression pattern of specific Wnts in this study using whole-kidney lysates at 5 weeks is quite different from that derived from the isolated glomeruli at 1 day after ADR injection,6 consistent with the notion that Wnt expression is dynamic and changes with times during renal injury.7 Regardless of what specific Wnt is induced, however, β-catenin, the common downstream mediator of the canonical Wnt signaling, is induced (Figures 6 and and8),8), indicating a robust activation of the canonical pathway of Wnt signaling in this model. Interestingly, this Wnt/β-catenin signaling is virtually blocked after paricalcitol treatment, underscoring that the vitamin D analog is able to constrain the activity of Wnt/β-catenin signaling in vivo.

Our results indicate that paricalcitol not only prevents the development of proteinuria after ADR injury but also induces reversal of an established proteinuria (Figure 8). These findings are quite significant and obviously have clinical relevance. It is conceivable that paricalcitol could inhibit Wnt/β-catenin signaling at least by two different mechanisms (Figure 9H). On one hand, paricalcitol selectively suppresses the expression of multiple Wnt genes including Wnt4, Wnt7a, Wnt7b, and Wnt10a, whose expression is up-regulated after ADR injury. This action presumably prevents Wnt induction and β-catenin activation in the injured kidney after ADR injection in the first place. On the other hand, even after β-catenin is activated in an established proteinuria, paricalcitol apparently has the ability to inhibit β-catenin–mediated gene transcription by inducing VDR binding to active, nuclear β-catenin (Figure 9). This leads to the sequestration of the β-catenin transcriptional activity in the nuclei. Such a mode of action of vitamin D analog not only occurs in glomerular podocytes but also in tubular epithelial cells (Figure 9), as well as in colon carcinoma cells.14 Of note, previous studies show that calcitriol (1,25-dihydroxyvitamin D3) inhibits Wnt signaling by inducing its antagonist DKK1 gene expression in a human colon cancer cell line.37 However, that mode of action is unlikely to be operative in the kidney, because paricalcitol does not induce the expression of any members of the DKK family in vivo (Figure 5).

Of many Wnt/β-catenin downstream target genes, Snail is well characterized and mostly relevant to proteinuria and renal fibrosis observed in ADR nephropathy.6,38 Recent studies suggest that podocytes also undergo EMT in response to injurious stimuli,39,40 in which Snail may play a role.41,42 Snail down-regulates key epithelial markers such as E-cadherin by binding to the E-box in the regulatory region of its target genes.4345 We have previously shown that β-catenin induces Snail expression in glomerular podocytes,6 which in turn directly suppresses the expression of nephrin.6,38,41 Overexpression of Snail is also sufficient to induce kidney injury and fibrosis, as illustrated in Snail transgenic mice.46 Therefore, Snail could be a major downstream effector of Wnt/β-catenin signaling that mediates podocyte dysfunction and tubular EMT by virtue of its ability to repress nephrin and E-cadherin expression (Figure 9H). Indeed, renal Snail expression is markedly induced after ADR injection, and paricalcitol substantially suppresses its induction. It is worthwhile to point out that Wnt signaling may influence Snail protein abundance by both transcriptional and post-translational mechanisms (Figure 7E), two distinct pathways regulated by β-catenin and glycogen synthase kinase-3β (GSK-3β), respectively. Although β-catenin directly induces Snail mRNA expression,6 Wnt-mediated GSK-3β inhibition results in Snail dephosphorylation, leading to its stabilization by preventing ubiquitin-mediated degradation.47,48 Consistent with this notion, the magnitude of Snail protein induction is greater than that of its mRNA after ADR injury (Figure 7).

The therapeutic efficacy of paricalcitol in ADR nephropathy is impressive, which could involve multiple mechanisms. In addition to modulating Wnt/β-catenin signaling, we cannot exclude the possibility that paricalcitol may elicit its beneficial activities by other routes as well. In that regard, paricalcitol has been shown to inhibit renal inflammation by promoting VDR-mediated sequestration of NF-κB signaling,49 consistent with a reduced renal infiltration of macrophages and decreased expression of proinflammatory cytokines in this study. Furthermore, paricalcitol is able to attenuate renal interstitial fibrosis by blocking tubular EMT,43,45 a process in which β-catenin and Snail play a critical role. Because renal inflammation and fibrosis are late-onset events, secondary to podocyte injury and proteinuria in this model, it is plausible that paricalcitol inhibition of Wnt/β-catenin signaling may play a primary and predominant role in protecting the kidney from developing ADR nephropathy. However, paricalcitol seems to inhibit β-catenin signaling after ADR injury in tubular epithelial cells as well (Figure 9), suggesting that some of its effects on the tubulointerstitium may be direct events.

We should point out that the signals controlling podocyte dysfunction, inflammation, and fibrosis may cross-talk with each other and are likely integrated in the path of Wnt/β-catenin/Snail signaling. Along this line, a recent study indicates that NF-κB activation leads to Snail stabilization by preventing its degradation, which links inflammation to the major product of Wnt/β-catenin signaling.50 Furthermore, Snail transcriptionally represses the expression of VDR,51 a potent inhibitor of Wnt/β-catenin signaling. This creates a vicious cycle of vitamin D deficiency, Wnt/β-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/β-catenin signaling and inhibits Snail expression, thereby preventing podocyte injury, proteinuria, and renal fibrosis in ADR nephropathy.

CONCISE METHODS

Animal Models

Mouse model of podocyte injury and proteinuria was established by intravenous injection of ADR, as described previously.6,25 Male BALB/c mice weighing 20–22 g were obtained from Harlan Sprague-Dawley (Indianapolis, Indiana). Three sets of animal experiments were performed. The first set consisted of three groups of mice: (1) normal control (n = 5); (2) ADR mice injected with vehicle (n = 8); and (3) ADR mice injected with paricalcitol (n = 8). ADR (doxorubicin hydrochloride; Sigma, St. Louis, Missouri) was administered by a single intravenous injection at 10 mg/kg body wt. Paricalcitol (kindly provided by Abbott Laboratories, Abbott Park, Illinois) was given by daily subcutaneous injection at 50 ng/kg body wt, starting at the time when ADR was administered. The dose of paricalcitol was chosen on the basis of our pilot experiments. At 5 weeks after ADR injection, all of the mice were sacrificed. The second set of experiments consisted of the same three groups as the first set, but the animals (n = 6) were sacrificed at 7 days after ADR injection. The third set of experiments consisted of five groups in which paricalcitol was administered either 1 day before or 2 and 6 days after ADR injection, respectively, and the animals were sacrificed at 3 weeks after ADR injection. The details of the experimental design for this set of experiments are presented in Figure 8A. Urine and kidney tissue were collected for various analyses. All of the animal studies were performed by use of the procedures approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh.

Urinary Albumin and Creatinine Assay

Urine albumin was measured by using a mouse albumin ELISA quantitation kit, according to the manufacturer's protocol (Bethyl Laboratories, Inc., Montgomery, Texas). Urine creatinine was determined by a routine procedure as described previously.52 Urinary proteins were also analyzed by SDS-PAGE after normalization to urinary creatinine. After separation by SDS-PAGE, urine proteins were stained with Coomassie Blue R-250.

Histology and Immunohistochemical Staining

Paraffin-embedded mouse kidney sections (3-μm thickness) were prepared by a routine procedure. The sections were stained with hematoxylin-eosin, periodic acid-Schiff reagent by standard protocol. Kidney sections were also subjected to Masson-trichrome staining for assessing collagen deposition and fibrotic lesions. Quantitation of the fibrotic area was carried out by a computer-aided morphometric analysis (MetaMorph; Universal Imaging Co., Downingtown, Pennsylvania), as described previously.43 Immunohistochemical staining was performed using a routine protocol.43 The antibodies used were as follows: affinity-purified anti-mouse F4/80 antigen (catalog number 14-4801; eBioscience, San Diego, California) and rabbit polyclonal anti-β-catenin antibody (ab15180; Abcam, Cambridge, Massachusetts).

Immunofluorescence Staining and Confocal Microscopy

Kidney cryosections were fixed with 3.7% paraformalin for 15 minutes at room temperature. After blocking with 10% donkey serum for 30 minutes, the slides were immunostained with primary antibodies against nephrin (catalog number 20R-NP002; Fitzgerald Industries International, Inc., Concord, Massachusetts), podocin (catalog number SC-22298), and WT1 (catalog number SC-192; Santa Cruz Biotechnology, Santa Cruz, California). The slides were viewed under a Leica TCS-SL confocal microscope.

Western Blot Analysis

Glomeruli were isolated by differential serving technique according to the method described elsewhere.53 The isolated glomeruli were lysed with radioimmune precipitation assay buffer containing 1% NP40, 0.1% SDS, 100 μg/ml PMSF, 1% protease inhibitor cocktail, and 1% phosphatase I and II inhibitor cocktail (Sigma) in PBS on ice. The supernatants were collected after centrifugation at 13,000 × g at 4°C for 20 minutes. Whole-kidney lysates were prepared using the same procedures. Cultured mouse podocytes were lysed in SDS sample buffer. Protein expression was analyzed by Western blot analysis as described previously.7 The primary antibodies used were as follows: anti-nephrin (Fitzgerald Industries International), anti-podocin, anti-WT1, anti-VDR (SC-1008), and anti-actin (SC-1616) (Santa Cruz Biotechnology), anti-β-catenin (catalog number 610154; BD Transduction Laboratories, San Jose, California), anti-α-SMA (clone 1A4; Sigma), anti-Snail (ab17732; Abcam), anti-TATA-binding protein (TBP) (catalog number ab181–100; Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Ambion, Austin, Texas).

Real-Time RT-PCR

Total RNA isolation and real-time RT-PCR were carried out by the procedures described previously.54 Briefly, the first strand cDNA synthesis was carried out by using a reverse transcription system kit according to the instructions of the manufacturer (Promega, Madison, Wisconsin). Real-time RT-PCR was performed on ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, California) as described previously.54 The PCR mixture in a 25-μl volume contained 12.5 μl of 2× SYBR Green PCR Master Mix (Applied Biosystems), 5 μl of diluted RT product (1:10), and 0.5 μm sense and antisense primer sets. The sequences of the primer pairs used in real-time PCR were given in Supplemental Table 1. PCR was run by using standard conditions. After sequential incubations at 50°C for 2 minutes and 95°C for 10 minutes, respectively, the amplification protocol consisted of 50 cycles of denaturing at 95°C for 15 seconds and annealing and extension at 60°C for 60 seconds. The standard curve was made from series dilutions of template cDNA. The mRNA levels of various genes were calculated after normalizing with β-actin. Expression of Wnt mRNA levels was determined as described previously.7

Cell Culture and Treatment

The conditionally immortalized mouse podocyte cell line was kindly provided by Dr. Peter Mundel (University of Miami, Miami, Florida), as described previously.41,55 The cells were cultured at 33°C in RPMI 1640 medium supplemented with 10% fetal bovine serum and recombinant IFN-γ (Invitrogen, Carlsbad, California). To induce differentiation, podocytes were grown under nonpermissive conditions at 37°C in the absence of IFN-γ. After serum starvation for 16 hours, the cells were treated with paricalcitol (10−7 m). Human proximal tubular epithelial cells (HKC, clone-8) were provided by Dr. L. Racusen (Johns Hopkins University, Baltimore, Maryland). Cell culture was 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.

Nuclear Protein Preparation

Nuclear protein preparation was carried out according to the procedure described previously.56 Briefly, mouse podocytes or HKC-8 cells after various treatments as indicated were washed twice with cold PBS and scraped off the plate with a rubber policeman. After centrifugation, the cell pellets were resuspended in Buffer A (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm 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 washed with Buffer B (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, and 1% protease inhibitor cocktail). The nuclei were lysed in SDS sample buffer. For loading control of nuclear protein, the blots were stripped and reprobed with antibody against the TBP.

Coimmunoprecipitation

Immunoprecipitation was carried out by using an established method.52 Briefly, mouse podocytes and HKC-8 cells after various treatments were lysed on ice in 1 ml of nondenaturing lysis buffer that contained 1% Triton X-100, 0.01 m Tris-HCl (pH 8.0), 0.14 m NaCl, 0.025% NaN3, 1% protease inhibitors cocktail, and 1% phosphatase inhibitors cocktail I and II (Sigma). After preclearing with normal IgG, cell lysates (0.5 mg of protein) were incubated overnight at 4°C with 4 μg of anti-VDR (Santa Cruz Biotechnology), followed by precipitation with 30 μl of protein A/G Plus-agarose for 1 h at 4°C. The precipitated complexes were separated on SDS-PAGE and immunoblotted with anti-β-catenin antibody.

Transfection and Luciferase Assay

The effect of paricalcitol on β-catenin–mediated gene transcription was assessed by using the TOP-flash TCF reporter plasmid containing two sets of three copies of the TCF binding site upstream of the thymidine kinase (TK) minimal promoter and luciferase open reading frame (Millipore, Billerica, Massachusetts). Podocytes were cotransfected by using Lipofectamine 2000 reagent (Invitrogen) with TOP-flash plasmid (1 μg) and VDR expression vector (1 μg) in the absence or presence of the stabilized β-catenin expression vector (pDel-β-cat). An internal control reporter plasmid (0.1 μg) Renilla reniformis luciferase driven under TK promoter (pRL-TK; Promega) was also cotransfected for normalizing the transfection efficiency. The transfected cells were incubated in serum-free medium without or with paricalcitol (10−7 m) as indicated. Luciferase assay was performed using a dual luciferase assay system kit according to the manufacturer's protocols (Promega). Relative luciferase activity (arbitrary units) was reported as fold induction over the controls after normalizing for transfection efficiency.

Statistical Analyses

Statistical analyses of the data were carried out using SigmaStat software (Jandel Scientific, San Rafael, California). Comparison between groups was made using one-way ANOVA followed by a Student-Newman-Kuel's test. P < 0.05 was considered significant.

DISCLOSURES

None.

Supplementary Material

Supplemental Data:

Acknowledgments

This work was supported by National Institutes of Health Grants DK061408, DK064005, and DK071040 and a Grant-in-Aid from the Abbott Laboratories. C.D. was supported by American Heart Association Beginning Grant-in-Aid 0865392D and the University of Pittsburgh Medical Center Health System Competitive Medical Research Fund.

Footnotes

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

Supplemental information for this article is available online at http://www.jasn.org/.

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