Primary cilia modulate balance of canonical and non-canonical Wnt signaling responses in the injured kidney

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Cilia; Epithelial; Fibrosis.

Figure 1

Renal expression of Wnt ligand genes upon unilateral ureter obstruction (A-K). We isolated total RNA from whole kidney tissues 3 and 7 days after ureter ligation and analyzed expression of putative Wnt ligand genes by quantitative real-time PCR. The bar graphs display relative mRNA expression of indicated Wnt ligand genes upon ureter ligation in relation to kidneys of sham-operated control mice. The arrows highlight robust expression of β-catenin in dilated tubules. Data is presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. no significance, UUO, unilateral ureteral obstruction; P values were calculated respective to control.

Figure 2

β-catenin immunolocalization in experimental obstructive nephropathy. We labeled kidneys of sham-operated mice and of mice which had been challenged with UUO with antibodies specific to (A) total β-catenin or with antibodies specific for (B) non-phosphorylated β-catenin (indicative of active canonical signaling). The pictures display representative photomicrographs of each groups, positive immunostaining is indicated by brown precipitate. The arrows highlight robust expression of β-catenin in dilated tubules. Scale bars: 100 μm. UUO, unilateral ureteral obstruction.

Figure 3

Relative expression of canonical and non-canonical Wnt target genes upon unilateral ureter obstruction. We isolated total RNA from whole kidneys of sham-operated control mice and of mice which had been challenged with UUO and analyzed expression of target genes of canonical Wnt signaling (Axin2, Wisp2, Trp53, and Ccnd1, graphs A-D) and of non-canonical Wnt target genes (CamK2a, Plcb1, Daam1, and Ptk7, graphs E-H). Data is presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. no significance; UUO, unilateral ureteral obstruction.

Figure 4

Stunted cilia and canonical Wnt signaling in experimental obstructive nephropathy. (A) Paraffin-embedded kidney sections were labeled with antibodies to acetylated α-tubulin (Ac-α-tubulin) (green). Nuclei were labeled with DAPI (blue). Staining was analyzed using a confocal microscope. The pictures display representative confocal photomicrographs of labeled sections of sham-operated mice (left) and of kidneys 3 days (middle) and 7 days (right) after ureter obstruction. Scale bars: 10 μm. Arrows indicate normal cilia and arrowheads indicate stunted cilia. (B) The graph summarizes the relative distribution of normal and stunted cilia in each group. (C) Immunofluorescence double-labeling experiments were performed using specific antibodies to acetylated α-tubulin (cilia) and non-phosphorylated β-catenin (active canonical Wnt signaling). Nuclei were stained with DAPI. The pictures display representative photomicrographs of sections from sham-operated control mice (left) and post-obstructive (days 3 and 7) kidneys. Non-phosphorylated β-catenin immunostaining was most abundant in tubules with stunted cilia. Arrows indicate normal cilia and arrowheads display stunted cilia. Scale bars: 10 μm. (D) The graph shows relative distribution of normal cilia and stunted cilia regarding to β-catenin expression in each group. (E) Non-fibrotic and fibrotic kidney sections from human biopsies were labeled with acetylated α-tubulin (cilia) and non-phosphorylated β-catenin. Pictures display representative confocal photomicrographs of each sample. Arrows indicate normal cilia and arrowheads indicate stunted cilia. Scale bars: 10 μm. UUO, unilateral ureteral obstruction; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 5

Reciprocal responses against Wnt in ciliated and non-ciliated cells. We compared induction of canonical and non-canonical Wnt target genes in response to Wnt3a in cultured ciliated and non-ciliated HK2 tubular epithelial cells. The graphs display relative mRNA expression of indicated genes in ciliated versus non-ciliated cells with or without Wnt3a in culture media. (A) Ciliated and non-ciliated HK2 cells were methanol fixed and were labeled with antibodies against acetylated α-tubulin (cilia) and non-phosphorylated β-catenin. Higher expression of active β-catenin was observed in Wnt3a-treated non-ciliated cells. Arrows highlight normal cilia on epithelial cell. Scale bars: 10 μm. (B-E) Expression of canonical Wnt signaling target genes (AXIN2, WISP2, TP53, CCND1) was substantially induced in ciliated cells but not in non-ciliated cells. (F-I) Addition of Wnt3a to culture media induced expression of non-canonical target genes in ciliated cells but not in non-ciliated HK2 cells (CAMK2A, PLCB1, DAAM1, PTK7). Data is presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. no significance, DAPI, 4′,6-diamidino-2-phenylindole. P values were calculated respective to each sample without treatment.