Primary cilia modulate balance of canonical and non-canonical Wnt signaling responses in the injured kidney
© Saito et al.; licensee BioMed Central. 2015
Received: 21 January 2015
Accepted: 20 March 2015
Published: 16 April 2015
While kidney injury is associated with re-expression of numerous Wnt ligands and receptors, molecular mechanisms which underlie regulation of distinct Wnt signaling pathways and ensuing biological consequences remain incompletely understood. Primary cilia are increasingly being recognized as cellular ‘antennae’ which sense and transduce signals from the microenvironment, particularly through Wnt signaling. Here, we explored the role of cilia as modulators of canonical and non-canonical Wnt signaling activities involving tubular epithelial cells in the injured kidney.
We demonstrate that in the mouse model of unilateral ureter obstruction, progression of kidney injury correlates with increased expression of numerous Wnt ligands, and that increased expression of Wnt ligands corresponded with over-activation of canonical Wnt signaling. In contrast, non-canonical Wnt signaling dropped significantly during the course of kidney injury despite gradually increased expression of typical non-canonical and intermediate Wnt signaling ligands. We further demonstrate that in cultured tubular epithelial cells, cilia modulate balance between canonical and non-canonical signaling responses upon exposure to Wnt ligands.
We provide evidence that in the context of renal injury, primary cilia act as molecular switches between canonical and non-canonical Wnt signaling activity, possibly determining between regenerative and pro-fibrotic effects of Wnt re-expression in the injured kidney.
KeywordsCilia Fibrosis Epithelial
In principle, the kidney possesses a unique capacity to repair itself, even upon severe acute injury [1-3]. In clinical reality, however, complete regeneration of acute kidney injury is not always achieved [4,5]. Furthermore, impaired regeneration is a principal feature of fibrosis, a pathological scarring process which is the basis of chronic progressive kidney disease [6-8]. In clinical nephrology, both acute kidney injury and chronic progressive kidney disease pose major problems, as therapies to halt fibrosis or to induce regeneration of acute, or even chronic kidney injury, are not yet available . Furthermore, the molecular mechanisms which discern between renal regeneration and chronic progressive fibrosis are still incompletely understood and insights derived from animal and cell culture studies have not yet been sufficiently translated into clinical context.
In this regard, Wnt signaling is being increasingly recognized as important mediator of both renal repair and progressive renal fibrosis. Wnt proteins (Wnt stands for Wingless-related integration site, a portmanteau of ‘int’ referring to the cancer-causing mammalian gene integration 1 and Wg referring to drosophila gene homolog known as wingless) in general are a family of secreted proteins, which act as ligands to activate distinct Wnt signaling pathways [10,11]. There are two principal Wnt-signaling pathways, the β-catenin-dependent ‘canonical’ Wnt pathway and the β-catenin-independent ‘non-canonical’ pathways . In mammalians, there are currently 19 known Wnt ligand proteins and 10 different Frizzled-Wnt receptors, and since there is no clear ligand-receptor-pathway relationship, mechanisms that discern between activation of distinct canonical and non-canonical pathways are complex and only incompletely understood .
Wnt signaling in general plays multiple essential roles in organogenesis, carcinogenesis, and tissue homeostasis . While in the kidney, Wnt-signaling is essential for nephron formation during kidney development, Wnt signaling activity is ominously silenced in the adult kidney . Injury of the adult kidney is associated with re-expression of numerous Wnt ligands and receptors . There are conflicting reports however, if such re-activation of Wnt signaling is beneficial and should be regarded as an attempt to re-activate developmental programs to repair the kidney [14-16] or if Wnt signaling is detrimental and contributes to progression of renal fibrogenesis [17-20].
While the role of Wnt signaling in the injured kidney is complex, majority of studies on canonical Wnt signaling demonstrate a detrimental role. A recent study implicated activity of β-catenin signaling - the prototypical read-out of canonical Wnt signaling - within tubular epithelial cells as a predictor of tubulointerstitial fibrosis and poor prognosis of kidney allografts . Majority of studies on non-canonical Wnt signaling however report a beneficial pro-regenerative role. Because previous studies demonstrated a predominantly beneficial role of non-canonical Wnt signaling, yet a pro-fibrotic activity of canonical Wnt signaling, we hypothesized that the shift from non-canonical to canonical Wnt signaling may contribute to the switch between physiological repair and pathological fibrogenesis in response to Wnt re-expression, and we aimed to gain further insights into the mechanisms which modulate Wnt signaling responses in the injured kidney.
In this regard, primary cilia have emerged as principal modulators of Wnt signaling . Primary cilia have been located on almost every mammalian cell type . Primary cilia have emerged as cellular ‘antennae’ which sense and transduce signals from the microenvironment, particularly through Wnt signaling . The kidney has emerged as the prime organ to study cilia biology as the most relevant genetic kidney diseases are caused by primary cilia defects (‘ciliopathies’). The role of cilia in kidney injury (in ‘nonciliopathies’) has not been studied in depth yet.
We observed that in the murine model of obstructive nephropathy irreversible tubulointerstitial fibrosis (7 days after ureter obstruction), co-incides with structural damage of primary cilia, overactivation of canonical Wnt signaling, and drop of non-canonical Wnt signaling activity. We provide causal evidence that loss of intact cilia contributes to the switch from non-canonical to canonical Wnt signaling in response to Wnt ligands by tubular epithelial cells, possibly contributing to the switch from reversible to irreversible kidney injury.
Activation of Wnt signaling in kidney fibrosis
Correlation of Wnt signaling switch and impairment of cilia
In summary, our studies revealed that progression of kidney injury upon ureter ligation is associated with increased canonical and decreased Wnt signaling and that such Wnt signaling switch is associated with increased Wnt ligand expression and progressive loss of cilia.
Here, we report two novel findings which add new aspects and possible explanation to the complexity of Wnt signaling in the context of kidney disease. While our study is in line with previous reports of increased canonical Wnt signaling during renal fibrogenesis (an intuitive finding in view of robust overexpression of multiple Wnt ligand genes), we report for the first time a distinct pattern of non-canonical Wnt signaling activity, as expression of non-canonical Wnt target genes drops during disease progression (despite increased expression of Wnt ligand genes). Second, we provide evidence for the first time that primary cilia (which deteriorate during progression of tubulointerstitial fibrosis) play a causal role in discerning between canonical and non-canonical Wnt signaling pathways, likely impacting the net-effect of Wnt activity in the injured kidney.
While several previous studies agree that kidney injury is associated with increased expression of Wnt ligands , their impact on disease progression has been controversial as some studies concluded that Wnt signaling contributes causally to progression of kidney fibrosis [17-20] whereas others reported that Wnt signaling is beneficial for the injured kidney [14-16]. However, most of these prior conflicting studies focused on canonical Wnt signaling, whereas the role of non-canonical Wnt signaling has been largely overlooked. Several studies reported however, that non-canonical Wnt signaling contributes causally to renal repair upon acute kidney injury, and hence, it is attractive to speculate that the sum effect of Wnt signaling in the injured kidney is at least in part determined by cilia-dependent non-canonical Wnt signaling. Context-dependent changes in the sum effect of Wnt signaling due to a switch from predominantly non-canonical to canonical Wnt signaling is not without precedent , and we believe that the intensity of non-canonical Wnt signaling is not the only determining factor in the context of kidney disease either . Such thinking may be further supported by our observation that cilia impairment and impaired non-canonical Wnt signaling canonical over-activation are only observed after 7 days of ureter ligation (when kidney injury is irreversible) but not yet after 3 days (when kidney injury is still reversible when the ligature is removed) .
With regard to conflicting interpretations of the sum effect of Wnt signaling on kidney fate, it may be also important to note that loss of cilia appears to be model specific and that we did not observe cilia stunting or β-catenin immune-labeling in other murine models of kidney disease (data not shown). Similarly, impact of genetic background (in our case C57BL/6 mice were used) remains unclear.
We are aware of the fact that the strongest clinical evidence for a detrimental role of canonical Wnt signaling stems from a study which analyzed utility of tubular β-catenin immunostaining as predictor of chronic allograft nephropathy . Of note, in this study, tubular β-catenin immunostaining was not analyzed as a read-out for canonical Wnt-signaling overactivation but as biomarker for epithelial-mesenchymal transition (EMT) . In the context of kidney disease, EMT (defined as acquisition of mesenchymal properties and loss of epithelia functions through induction of specific EMT-transcriptional programs) is considered to contribute causally to progression of tubulointerstitial fibrosis through impairment of tubular functions and also through contribution to interstitial fibroblast accumulation [6,42]. In this regard, Wnt-initiated β-catenin signaling was identified as original EMT pathway in palate formation [43,44], and numerous studies corroborated β-catenin signaling as the master regulator of EMT in the context of embryonic development, cancer progression, and organ fibrosis [45-47]. Furthermore, previous studies linked Wnt-induced EMT to p53 accumulation, and our studies demonstrate increased p53 mRNA expression in response to cilia impairment [48,49]. Hence, one may speculate that loss of cilia in injured tubular epithelial cells contributes to progression of chronic kidney disease through facilitating a switch from non-canonical to canonical Wnt signaling, ultimately contributing to EMT of affected epithelial cells.
Because cilia play a central role in pathogenesis of hereditary cystic kidney diseases (because most mutations causing hereditary cystic kidney diseases affect genes which encode for cilia proteins), it is tempting to compare our observations with existing polycystic kidney disease (PKD) literature. In this regard, increased Wnt signaling per se is considered to contribute causally to manifestation of autosomal dominant polycystic kidney disease . Cystogenesis associated with nephronophthisis (NPH), which is caused by mutations of genes encoding for nephrocystin proteins (NPHPs) is considered to be driven by over-activation of canonical Wnt signaling, and NPHP4 causally inhibits canonical Wnt signaling through enabling proteolytic degradation of β-catenin . In this context, it is attractive to speculate that observed cilia loss and β-catenin over-activation in cystically dilated tubules in the UUO model contribute to progression of tubular atrophy through mechanisms which are similar to those which contribute to cystogenesis in PKD. While such studies are beyond the scope of this study, qualitative analysis of stunted cilia in the UUO model may provide important insights into the role of cilia in modulation of Wnt signaling and progression of chronic kidney disease in the future.
We are aware that our study focuses entirely on the impact of cilia and Wnt signaling in tubular epithelial cells. If such mechanism is relevant for other cell types such as fibroblasts remains to be seen.
Primary cilia favor non-canonical Wnt signaling responses in tubular epithelial cells, while loss of intact cilia augments canonical Wnt signaling in response to Wnt ligands. As β-catenin superactivation has been identified as biomarker for negative outcome of chronic kidney disease and kidney injury is associated with increased expression of most Wnt ligands, impairment of primary cilia likely contributes causally to renal fibrogenesis by serving as molecular switch from non-canonical to canonical Wnt signaling responses.
Unilateral ureteral obstruction
Eight- to twelve-week-old CD57BL/6 mice were anesthetized with isoflurane inhalation. Analgesia was performed by subcutaneous Buprenorphine injection. The ureter was separated from the surrounding tissues, and two ligatures were placed about 5 mm apart in upper two thirds of the ureter of the left kidney to obtain reliable obstruction. Mice were sacrificed at indicated time points after ureter ligation . All studies had been carried out with the approval of local authorities (Tierschutzkommission Universitätsmedizin Göttingen) and LAVES (Landesamtes für Verbraucherschutz und Lebensmittelsicherheit; G 12/685).
Fomalin-fixed, paraffin-embedded kidney sections from mice and human biopsy samples were deparaffinized in xylene and dehydrated through graded alcohols. Antigen retrieval was undertaken in a steam cooker for 40 min in Target retrieval solution (Dako, Glostrup, Denmark). Endogenous peroxidase was blocked with 3% hydrogen peroxide (H2O2) in methanol for 30 min. Samples were blocked with 2.5% horse serum (Dako, Glostrup, Denmark). After the blocking step, sections were incubated with primary antibody for overnight at 4°C in a humidified chamber, and the biotinylated secondary antibody (Dako, Glostrup, Denmark) was applied for 30 min at room temperature. The reaction products were visualized using 3,3′-diaminobenzidinetetrahydrochloride (DAB) (Dako, Glostrup, Denmark) and slides were counterstaining with hematoxylin. Mouse monoclonal β-catenin antibody (sc-7963: Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and rabbit monoclonal non-phospho (Active) β-catenin antibody (#8814: Cell Signaling, Danvers, MA, USA) were used as primary antibodies.
Immunofluorescence staining of cells was performed in chambered slides (BD Bioscience, San Jose, CA, USA). Cells were seeded in chambered slides and fixed in ice-cold methanol for 10 min. Slides were then washed in phosphate-buffered saline (PBS) three times, and they were incubated with blocking solution (3% BSA in PBS) for 30 min. For immunofluorescence staining of mouse tissues, formalin-fixed, paraffin-embedded sections were used. Slides were deparaffinized and dehydrated. Mouse monoclonal acetylated α-tubulin antibody (Sigma-Aldrich, St. Louis, MO, USA) and non-phospho β-catenin antibody (Cell signaling, Danvers, MA, USA) was used as primary antibodies. Samples were then stained with Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 568-conjugated anti-rabbit IgG antibody (Life technologies, Carlsbad, CA, USA) and mounted with VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, CA, USA) with DAPI (4′,6-diamidino-2-phenylindole).
Confocal microscopy and image analysis
Cells were viewed by using the Axiovert-200 inverted light and epifluorescence microscope (Carl Zeiss, Oberkochen, Germany) or using the confocal LSM 780 microscope (Carl Zeiss, Oberkochen, Germany). Images of cilia were captured from randomly chosen high-power fields (40× objective), when the full extent of cilia could be visualized in a single plane of focus. Cilia were scored as either possessing a full length primary cilia (>2 μm) or possessing a stunted primary cilia (<2 μm). Cilia from ten proximal and ten distal tubules were measured for each mouse.
HK2 human proximal tubular epithelial cells were cultured in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 100 g/mL penicillin, 100 g/mL streptomycin, and 10% fetal bovine serum (FBS, Cellgro, Manassas, VA, USA) at 37°C in 5% CO2. For immunostaining of cilia in culture, cells are grown as a monolayer on chambered slides (BD Bioscience, San Jose, CA, USA) and for RNA isolation in six-well plates (Greiner Bio-One, Kremsmunster, Austria). To generate ciliated and non-ciliated tubular epithelial cells, we seeded HK2 cells at different densities (six-well plates; 5,000 vs 30,000 per well in six-well plates) and starved cells for 48 h without serum to induce cell cycle exit and ciliogenesis as described previously . Upon serum starvation, we exchanged media with growth factor-free control media or with media supplemented with 100 ng/ml recombinant Wnt3a (R&D systems, Minneapolis, MN, USA) for 48 h.
Tissue and cells were dissolved in TRIZOL (Invitrogen, Carlsbad, CA, USA), and tissue were shredded using TissueLyser LT (Qiagen, Hilden, Germany). RNA was isolated by using PureLink RNA Mini Kit (Ambion, Carlsbad, CA, USA) according to the manufacture’s protocol.
Quantative real-time PCR quantification
For SYBR-based real-time PCR, cDNA synthesis was performed by using DNase I (Sigma-Aldrich, St. Louis, MO, USA) treatment and SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Reverse-transcripted cDNA was added to the reaction mixture containing the primer pair and diluted with Fast SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA). PCR reactions were performed in a 96-well reaction plate using the StepOne Real-Time System (Applied Biosystems, Carlsbad, CA, USA) and were done in triplicates. All qRT-PCR data for RNA expression analysis were calculated using the ΔΔCT method. For statistical evaluation, t-test or a one-way ANOVA was used. Oligonucleotide sequences are shown in Additional file 2.
The use of parts of kidney biopsies for research purposes was approved by the Ethics Committee of the University Medical Center Göttingen, and written consent was obtained from all subjects before kidney biopsy. Detailed clinical patient data are presented in Additional file 1.
Bovine serum albumin
Calcium/calmodulin-dependent protein kinase type II alpha chain
Disheveled-associated activator of morphogenesis 1
Glyceraldehyde 3-phosphate dehydrogenase
Landesamtes für Verbraucherschutz und Lebensmittelsicherheit
Polycystic kidney disease
Tyrosin protein kinase-like 7
Real-time-polymerase chain reaction
Tumor protein p53
Tumor protein p53
Unilateral ureteral obstruction
Wnt1 inducible signaling pathway protein 2
This work was in part supported by grants from the Deutsche Forschungsgemeinschaft (ZE523/2-1 and ZE523/3-1 to MZ and equipment grant INST1525/16-1 FUGG) and funds from the University of Göttingen Medical Center (UMG).
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