Sclt1 deficiency causes cystic kidney by activating ERK and STAT3 signaling

Abstract

Ciliopathies form a group of inherited disorders sharing several clinical manifestations because of abnormal cilia formation or function, and few treatments have been successful against these disorders. Here, we report a mouse model with mutated Sclt1 gene, which encodes a centriole distal appendage protein important for ciliogenesis. Sodium channel and clathrin linker 1 (SCLT1) mutations were associated with the oral-facial-digital syndrome (OFD), an autosomal recessive ciliopathy. The Sclt1^–/– mice exhibit typical ciliopathy phenotypes, including cystic kidney, cleft palate and polydactyly. Sclt1-loss decreases the number of cilia in kidney; increases proliferation and apoptosis of renal tubule epithelial cells; elevates protein kinase A, extracellular signal-regulated kinases, SMAD and signal transducer and activator of transcription 3 (STAT3) pathways; and enhances pro-inflammation and pro-fibrosis pathways with disease progression. Embryonic kidney cyst formation of Sclt1^–/– mice was effectively reduced by an anti-STAT3 treatment using pyrimethamine. Overall, we reported a new mouse model for the OFD; and our data suggest that STAT3 inhibition may be a promising treatment for SCLT1-associated cystic kidney.

Introduction

A primary cilium is a microtubule-based organelle that protrudes from the basal body and emanates from the surface of many types of eukaryotic cells (1,2). The cilium is assembled from the distal end of a mother centriole in the G0/G1 phase of the cell cycle, by axonemal extension from the centrosome at the cell surface into the ciliary lumen. The cilium is disassembled before cells enter mitosis (3). Long thought of as a vestige of evolution, primary cilia are now considered as important sensory antennae that detect and transmit signals from the environment to the cell body in order to regulate embryonic development and postnatal tissue homeostasis (4). Defects in the formation or function of cilia cause a broad spectrum of human diseases known as ciliopathies, including Bardet-Biedl syndrome, oral-facial-digital syndrome (OFD) and polycystic kidney disease (5–7). The ciliopathies have common clinical features including retinitis pigmentosa, cystic kidney and polydactyly. Additional manifestations include hepatic disease, diabetes, situs inversus, cognitive disease and skeletal dysplasia (8–10).

Polycystic kidney disease (PKD) is the most common genetic disorder leading to renal failure and death in humans. It is characterized by kidney enlargement and loss of renal function because of the accumulation of numerous fluid-filled cysts (11). In PKD, the cysts originate from the renal tubules because of abnormalities in cell proliferation, fluid secretion, extracellular matrix and differentiation (12). PKD can be subcategorized as autosomal recessive PKD (ARPKD) or autosomal dominant PKD (ADPKD) (13–15). ARPKD is very rare, with an incidence of 1:20 000–40 000, and has a very early onset, i.e. in neonates and children. It is often caused by mutations of the PKHD1 gene, which encodes fibrocystin/polyductin (13–15). In contrast, the autosomal dominant form, ADPKD, is much more common, with an incidence of 1:500–1000 in the adults; it initiates in the fetus and grows continuously throughout an individual’s lifetime, ADPKD is caused by mutations of the PKD1 gene (85%), which encodes polycystin-1 (PC1), or the PKD2 gene (15%), encoding polycystin-2 (PC2). PC1 is a large integral membrane protein and has been proposed to function as a G protein-coupled receptor (16). PC2 is a cation-selective ion channel with a specificity for calcium (16). PC1 and PC2 form a heterodimer in the primary cilium. It was reported that the large PC1 extracellular domain senses mechanical cues such as flow stress; it triggers the PC2 cation channel to allow calcium influx, thereby activating calcium-dependent pathways (17,18). However, the ability of PC1/PC2 to allow calcium influx into the cilium is still controversial, as other studies have suggested that PKD caused by loss of polycystins may be not because of defects in mechanically induced, cilia-initiated calcium signaling (19,20). In ADPKD, calcium influx is low, and cyclic adenosine monophosphate (cAMP)/ protein kinase A (PKA) signaling is enhanced (21). This is thought to activate extracellular signal-regulated kinases (ERK)-mediated proliferation, to stimulate chloride and fluid secretion, and to promote signal transducer and activator of transcription 3 (STAT3)-induced chemokine and cytokine expression (21). In addition to PKHD1, PKD1 and PKD2, more than 40 genes have been found to be associated with cystic kidney (8), and new genetic causes of cystic kidney are still being discovered.

Sodium channel and clathrin linker 1 (SCLT1) was initially reported as an adapter protein that links sodium voltage-gated channel alpha subunit 10 (SCN10A) to clathrin (22). More recently, it has been identified as one of the five proteins that form centriole distal appendages, which facilitate the docking of the mother centriole to the plasma membrane to promote cilia initiation (23). A deficiency in SCLT1 in cultured retina pigmented epithelium cells markedly hampers ciliogenesis (23). SCLT1 mutations have been found in patients with OFD syndrome Type IX, a rare genetic disease characterized by midline cleft, microcephaly and colobomatous microphthalmia/anophthalmia. OFD patients may also exhibit polydactyly, absent pituitary gland, congenital heart disease and cystic liver and kidneys (24,25).

Here, we identified and characterized transgenic mouse line with an insertional mutation in Sclt1. Homozygous mutant mice exhibit cystic kidneys and other ciliopathy phenotypes. We also showed that inhibition of STAT3 signaling can ameliorate the formation of the kidney cysts in Sclt1^–/– mice.

Results

Sclt1 mutation caused cystic kidneys and polydactyly

By inbreeding the transgenic mouse line CAGGS-SB10 (26), we observed a distinctive preaxial polydactyly of the hindlimbs in 25% of the offspring, suggesting that the transgenic insertion event caused a recessive autosomal mutation affecting limb development (Fig. 1A). We named this mouse line Two Thumbs (TT). Despite the polydactyly, the newborn TT mice were typically viable and had normal body size, but their weight gain was slower than wild-type (WT) littermates (Fig. 1B). Most of the mutant mice showed a severely wasting phenotype and died before 1 month of age. To identify the genetic basis of these phenotypes, we first localized the transgene integration site to chromosome 3B by fluorescence in situ hybridization (FISH) (Fig. 1C). By candidate gene RT-PCR using total RNA from the mutant kidneys, we found that Sclt1 gene expression was disrupted, while expression of the two flanking genes, Jade1 and D3Ertd751e, was normal (Fig. 1D). Subsequent RT-PCR and genomic PCR walking experiments revealed that the transgene integrated within intron 12 of the Sclt1 gene (Fig. 1D), causing a 3-kb deletion, but not affecting the two adjacent exons (Supplementary Material, Fig. S1A–C). By RNA-seq, we found that the TT kidney completely lost the transcripts containing Sclt1 exons 13–21, which code the amino acids (a.a.) 350–688 of the C-terminal. That area comprises three major functional domains: MTD (myosin tail domain, a.a. 250–440), V-pump (vacuolar ATPase homology domain, a.a. 441–540) and the CTD (C-terminal domain containing multiple di-leucine motifs, a.a. 541–688) (Supplementary Material, Fig. S2). Moreover, because of the typically autosomal-recessive genetic pattern, this truncation of the SCLT1 protein is likely to cause a loss of function or severe homomorphism, instead of hypermorphism or neomorphism. Thus, in the rest of the text, we refer to the TT mice as Sclt1^–/– mice.

Figure 1

 Sclt1 deficiency caused preaxial polydactyly and cystic kidney. (A) Polydactyly phenotype of E13.5 and P1 (right) TT mice. (B) Growth curves of WT and TT mice at P1, P8 and P21 (n = 4, *P<0.05, error bars: standard deviations). (C) A single transgene insertion site was detected by FISH to mitotic chromosomes from a homozygous transgenic mouse (left, yellow arrows); the karyotype of mouse chromosome 3 is shown on the right (the black arrow indicates the region of hybridization). (D) Schematic depiction of the integration site (upper). The transgene (TG) inserted into intron 12 of the Sclt1 gene. Sclt1 transcripts did not amplify from kidney RNA of TT mice, while the two flanking genes, Jade1 and D3Ert757e, were normally expressed (lower). (E) The kidneys of Sclt1^–/– mice were enlarged and pale at p8 and p21. (F) Renal cysts were present in Sclt1^–/– mice at P1, P8 and P21 (scale bar = 500 μm). (G) Dramatically decreased length of the gut in the Sclt1^–/– mice. (H)Sclt1^–/– mice were often born with cleft palate (blue arrow).

Autopsies on the mutant mice revealed a distinctive, early onset, cystic kidney phenotype. The kidney weight and kidney index (KI = kidney weight/body weight × 100) of the mutants were slightly increased at postnatal day 1 (P1) (Supplementary Material, Fig. S3A). By P8, Sclt1^–/– mice showed growth retardation, and their kidneys were moderately larger and paler than WT kidneys (Fig. 1E). By P21, the KO kidneys were more than three times larger than those of the wild type (Fig. 1E and Supplementary Material, Fig. S3A). Histological analyses of kidneys from P1, P8 and P21 mice further showed the early onset and rapid progression of renal cysts, which were initiated from tubules in the medulla and then gradually expanded to the whole kidney (Fig. 1F). Furthermore, mutant kidneys at P21 displayed dramatically decreased cellularity in the cyst-lining epithelium (Fig. 1F). To identify the tubular origin of cysts, we co-stained with Dolichos biflorus agglutinin (DBA, for the collecting duct) and Lotus tetragonolobus lectin (LTL, for the proximal tubule) on P1 kidney sections; we found that the cysts originated from both the collecting and proximal tubule non-selectively (Supplementary Material, Fig. S4).

Beyond the kidney phenotype, we observed several developmental abnormalities caused by Sclt1 loss. The length of the intestine was significantly shorter in Sclt1^–/– mice than in WT mice (Fig. 1G and Supplementary Material, Fig. S3C), suggesting that Sclt1 also plays a role in gut development. In addition, we observed a moderate frequency of cleft palate in Sclt1^–/– mice (Fig. 1H). Moreover, relative to WT mice, Sclt1^–/– mice showed significant serum BUN (blood urea nitrogen) (Supplementary Material, Fig. S5), suggest a failure in renal function, which together with the short intestine and cleft plate may contribute to the high death rate of these mice.

Sclt1 deletion led to defective ciliogenesis, as well as increased cell proliferation and apoptosis

SCLT1 is a component of the centriole distal appendage, which promotes membrane docking and is necessary for ciliogenesis (23). Both the polydactyly and cystic kidney phenotypes found in Sclt1^–/– mice are hallmarks of ciliopathies. Therefore, we next asked whether loss of Sclt1 leads to defective cilia formation in the kidney. We observed that relative to wild-type (WT) mice, Sclt1^–/– mice had impaired cilia formation in the kidney, especially in the tubules. Based on our observation, we conclude that Sclt1^–/– mice have complete cilia loss (not a shortening) in the renal cells (Fig. 2A). GFP-tagged SCLT1 primarily localized to the vesicles in peri-centrosome/basal body regions of NIH3T3 cells (Fig. 2B).

Figure 2

Ciliogenesis defects in the kidney, and peri-centrosomal localization of GFP-SCLT1. (A)Sclt1^–/– mice show fewer cilia in the renal tubule epithelial cells. Green, acetylated tubulin antibody staining for the primary cilia; blue, DAPI staining for nuclei; yellow arrows denote the cilia-rich areas in the WT samples (scale bar = 50 μm). (B) Plasmid-expressed GFP-SCLT1 (green) localized in the peri-centrosomal vesicles of NIH3T3 cells. γ-Tubulin (red) was used as a marker for centrosomes. White arrows indicate the pericentrosomal regions in the two cells that express GFP-SCLT1 (scale bar = 50 μm).

To explore the cellular mechanisms by which Sclt1 loss causes cyst formation, we first assessed cell proliferation, which is considered an important factor in the development of renal cysts in PKD (27). In WT mice, we found a strong staining for Ki67 (a cell proliferation marker) at P1, consistent with previous reports that active cell proliferation is required for proper neonatal kidney development (28). However, P1 Sclt1^–/– mice showed an even higher Ki67 expression in both the renal cortex and medulla. In contrast to P1, WT kidneys showed overall decreased Ki67 expression, while Sclt1^–/– kidneys maintained a high percentage of Ki67-positive cells at P8 and P21 (Fig. 3A and B).

Figure 3

Sclt1 deficiency led to increased proliferation and apoptosis. (A) Compared with WT controls, Sclt1^–/– kidney sections showed an increased proportion of Ki67-positive cells at P1, P8 and P21. The medullary areas (yellow rectangles) and the cortical areas (red rectangles) are enlarged and presented in the second and third panels, respectively (scale bar = 50 μm). (B) Quantification of Ki67-positive cells in WT and Sclt1^–/– kidney at P1, P8 and P21 (n = 3, *P<0.05, error bars: standard deviations). (C and D)Sclt1^–/– kidneys had increased cleaved-caspase3-positive cells at P8 and P21 (C) and increased protein expression at P21 (D) (scale bar = 50 μm).

Despite that higher cell proliferation, mutant kidneys displayed dramatically the observed decreased cellularity in the cyst-lining epithelium at P21 (see Fig. 1F), suggesting that apoptosis may also contribute to the overall cystic phenotype. Indeed, in the Sclt1^–/– kidney sections, we also observed a significantly increased number of cells positive for cleaved caspase3, a well-defined marker for apoptotic cells (Fig. 3C). This was confirmed by western blot using whole-kidney lysates (Fig. 3D). Overall, these data suggest that the enhanced cell proliferation and apoptosis contribute to the development and progression of cystic kidney caused by Sclt1 loss.

Pathway analysis of the Sclt1-loss altered gene expression profiles

To further unbiasedly identify the signaling pathways responsible for Sclt1-loss-caused cystic kidney phenotype, we isolated mRNA from the whole kidneys of P1 Sclt1^–/– versus WT mice for RNA sequencing (RNA-seq). Gene set enrichment analyses (GSEA) on these RNA-seq data indicated that the Xenobiotic metabolism pathway (P = 0, FDR = 0.007) is significantly enhanced, implicating that the cystic kidney phenotype interrupted the detoxification function of kidney. Moreover, the interferon alpha response pathway (P = 0.0067, FDR = 0.044), IL6-JAK-STAT3 pathway (P = 0.072, FDR = 0.25), cell-cycle check point (P = 0.042, FDR = 0.25) and mitotic spindle pathway (P = 0.075, FDR = 0.25) are among the top most highly enriched gene sets in the Sclt1 loss kidneys (Supplementary Material, Fig. S6A and B). These data provided guidance for us to explore the related signaling mechanisms downstream of SCLT1 in kidney as described later.

Sclt1 loss activated cAMP/PKA/ERK signaling

Ciliary bending caused by flow-shear stresses normally increases the Ca²⁺ concentration in kidney cells and suppresses cAMP/PKA signaling (29,30). Ciliogenesis defects commonly cause enhanced PKA signaling, which can activate mitogenic ERK signaling to promote proliferation (31–33). This is in line with our histological observation and RNA-seq data that mitogenesis and cell cycle related pathways were enhanced (Fig. 3A and B and Supplementary Material, S3). Moreover, ERK signaling can also promote apoptosis, which was elevated in the Sclt1^–/– kidney as well (Fig. 3C and D). These motivated us to assess the changes of ERK signaling. By western blots, markedly higher p-ERK1/2 levels were detected in the whole kidney lysates from Sclt1^–/–mice in contrast to WT controls (Fig. 4A). We also observed that a subset of cyst-lining epithelial cells from the mutant kidneys showed a dramatic increase of nuclear p-ERK1/2 (Fig. 4B). In addition, the expression of downstream target genes of ERK signaling, such as Fosb and Fos, was significantly elevated in Sclt1^–/– mouse kidneys (Supplementary Material, Fig. S7). The activities of ERK and Jun luciferase reporters also were both dramatically increased in HEK293 human embryonic kidney cells with shRNA-mediated Sclt1 knockdown (SCLT1^KD) (Fig. 4C).

Figure 4

Sclt1 loss activated the ERK and cAMP/PKA signaling pathways. Sclt1^–/– kidneys showed higher p-ERK1/2 levels at P1, P8 and P21 by western blot (A) and by immunohistochemistry (B) (scale bar = 50 μm). (C)SCLT1^KD HEK293 cells showed enhanced luciferase expression from ERK and Jun reporters compared with control cells. (n = 3, *P < 0.05; error bars: standard deviation). (D) Nearly all P21 Sclt1^–/– kidney cells stained positive for p-CREB (scale bar = 50 μm). (E)SCLT1^KD HEK293 cells showed higher luciferase expression from a PKA/CREB reporter than the control cells. (n = 3, *P < 0.05; error bars: standard deviation). (F) SCLT1 and PC2 can be co-immunoprecipitated. HEK293 cells co-transfected with the V5-SCLT1 and Flag-PC2 plasmid were used for immunoprecipitation using anti-FLAG or anti-V5 (right) antibody. (G) PC2 overexpression reduced the Sclt1-loss-induced enhancement of ERK, Jun, and CREB luciferase reporters (n = 3, *P < 0.05; error bars: standard deviation).

ERK can be phosphorylated by the upstream Raf kinases, which can be regulated by protein kinase A (PKA) (34,35). Hence, we tested whether the hyperactivated ERK signaling in Sclt1-deficient cells was accompanied by elevated cAMP/PKA signaling. We detected a dramatic increase of nuclear p-CREB (cAMP response element binding protein) in the kidneys of Sclt1^–/– mice (Fig. 4D). Moreover, a cAMP response element/CREB-luciferase reporter (36) showed dramatically higher activity in the SCLT1^KD cells than in control HEK293 cells, suggesting that PKA signaling can be suppressed by SCLT1 (Fig. 4E).

A recent proteomic study showed that PC2 and SCLT1 are found within the exocyst complex (37). By co-immunoprecipitation, we validated that SCLT1 can interact with PC2 (Fig. 4F). Because the PC2/PC1 complex modulates calcium signaling in the cilia, we next tested whether PC2 overexpression could inhibit Sclt1-loss-induced ERK and PKA activation. We found that a gain of PC2 reduced the activation of the PKA and ERK pathways, as indicated by the reduction in ERK-, Jun-, and CRE/CREB-luciferase reporter expression in the SCLT1^KD cells (Fig. 4G). These data suggest that Sclt1 loss acts through the PC2/cAMP/PKA signaling axis to promote ERK signaling.

Sclt1 loss activated the STAT3 and TGF-β/SMAD signaling pathways

Our GSEA of RNA-seq data suggest that the interferon alpha response pathway (P = 0.0067, FDR = 0.044) and IL6-JAK-STAT3 pathway (P = 0.072, FDR = 0.25) were among most enhanced pathways in the Sclt1^−/− kidney compared with WT controls. It is worthy to note that these two pathways are highly correlated since interferon alpha signaling can activate JAK-STAT3 signaling (38,39). Moreover, PKA signaling was reported to activate STAT3, which further induces the transcription of cytokines, chemokines, and growth factors (40). Therefore, we next assessed the changes of STAT3 signaling in the kidneys. In WT mice, the levels of p-STAT3 (Y 705) were present during kidney development and growth but dwindled to a very low level in adult kidneys (41). In contrast, Sclt1^–/– kidneys showed a consistently higher level of pSTAT3 than did WT kidneys at all ages tested (P1, P8 and P21) (Fig. 5A). From IHC data, nuclear p-STAT3 was dramatically increased in the mutant kidney cyst-lining cells (Fig. 5B). Moreover, expression of STAT3 downstream cytokines and chemokines, including Il1α, Il1β, Il6 and Ccl2, was significantly increased in the Sclt1^–/– kidney (Fig. 5C), implying that Sclt1 loss may augment renal inflammation, which is considered one of the driving causes of ADPKD progression (42).

Figure 5

Sclt1 loss activated STAT3. Sclt1^–/– kidney lysates had increased p-STAT3 levels at P1, P8 and P21 by western blot (A) and by immunohistochemistry (B) (scale bar = 50 μm). (C) qRT-PCR shows that expression of STAT3 downstream genes was increased in the P21 Sclt1^–/–kidney compared with WT controls. Expression changes are normalized to actin as a control and presented in a log2 scale (n = 4; *P < 0.05; error bars: standard deviations).

Another key aspect of PKD progression is fibrosis, which has been identified as a significant manifestation of end-stage renal disease. We examined several fibrosis markers in the kidneys of P21 Sclt1^–/– mice and found a dramatic increase of plasminogen activator inhibitor-1 (Pai-1) and connective tissue growth factor (Ctgf) mRNA relative to WT kidney (Fig. 6A). PAI-1 is a potent fibrosis-promoting glycoprotein frequently induced by injury (43), and CTGF is a key mediator of tissue remodeling, wound healing, and fibrosis (44). Furthermore, increased expression of several extracellular matrix (ECM) components, including fibronectin and types I and type III collagen, was detected in the Sclt1^–/– kidney (Fig. 6A). We also found significantly increased expression of the ECM remodelers Mmp3. Mmp14 and Timp1 (Fig. 6A). The expression of Mmp9 and Mmp13 was decreased (Fig. 6A).

Figure 6

Sclt1 loss caused fibrosis and activated TGF-β/SMAD signaling. (A) qRT-PCR assays show that expression of fibrosis-associated genes was increased in the P21 Sclt1^–/– kidney compared with WT controls. Expression changes are normalized to actin as a control and presented in a log2 scale (n = 4; *P < 0.05; error bars: standard deviations). (B) Enhanced TGFβ signaling (shown by elevated p-SMAD2/3 levels) and unchanged BMP signaling (shown by p-SMAD1/5/8) in P21 Sclt1^–/– kidneys. (C) Marked increase in p-SMAD2/3-positive cells in the tubular epithelium of P21 Sclt1^–/– kidney by immunohistochemistry (scale bar = 50 μm). (D) Luciferase expression from a TGFβ reporter (Smad binding element [SBE]) was increased in SCLT1^KD HEK293 cells, but was unchanged from the ID1 promoter (n = 3; *P < 0.05; NS, no statistical significance; error bars: standard deviations).

Because Pai1 and Ctgf are among the most robust target genes of the TGF-β/SMAD signaling pathway, which is the master regulator of fibrosis (45), we assessed whether the TGF-β/SMAD signaling pathway was altered by Sclt1 loss. The Sclt1^–/– kidneys showed a dramatic increase of p-SMAD2/3 (TGF-β signaling) but a slight decrease of p-SMAD1/5/8 (BMP signaling) (Fig. 6B). Consistent with this, p-SMAD2/3 nuclear localization was increased in the epithelial cells of cystic tubules of Sclt1^–/– kidneys (Fig. 6C). In addition, compared with control cells, the SCLT1^KD HEK293 cells showed elevated activity of an SBE-luciferase reporter (Smad2/3 binding element for TGF-β signaling), but no changes in the activity of an Id1-luciferase reporter (for BMP signaling) (Fig. 6D). Our data suggest that Sclt1 loss activated the TGF-β/SMAD and STAT3 signaling pathways, causing fibrosis and inflammation to promote PKD progression.

Pyrimethamine suppressed cyst formation and growth in Sclt1^–/– kidneys

A recent study reported that pyrimethamine, originally used as an anti-parasitic compound and specifically inhibits STAT3 tyrosine phosphorylation and transcriptional function, can effectively suppress kidney cyst formation in Pkd1-deficient mice (46). This suggested that pyrimethamine treatment might alleviate cystogenesis in Sclt1^–/– mice. To test this, we treated E12.5 embryos with pyrimethamine (30 mg/kg), using DMSO as a control, by daily gavage to pregnant female mice for 7 day. Compared with DMSO-treated mutant mice, pyrimethamine-treated mutant mice had significantly reduced kidney weight and kidney/body weight ratio, as well as an increased gut length, without a significant change in body weight or other signs of toxicity (Supplementary Material, Fig. S8A–D). By histology, the pyrimethamine-treated mice had significantly fewer and smaller cysts and thus a decreased cystic index (Fig. 7A). Furthermore pyrimethamine treatment alleviated the overproliferation of Sclt1^–/– kidney cells, as indicated by fewer Ki67-positive cells (Fig. 7B andSupplementary Material, S8E). However, the treatment did not affect apoptosis in either WT or Sclt1^–/– kidneys, based on the unchanged cleaved caspase3 staining (Fig. 7C).

Figure 7

Pyrimethamine suppressed cyst formation in Sclt1^–/– kidneys. Pyrimethamine treatment decreased cyst formation, as shown by the number of cysts and cyst index (cyst index = cyst area/total area × 100, n = 3, *P < 0.05, error bar: standard deviations) (A) (scale bar = 100 μm), and reduced cell proliferation as indicated by Ki67 IHC (B) (scale bar = 50 μm), but did not change apoptosis as indicated by cleaved caspase3 IHC at P1 (C) (scale bar = 50 μm). Mutant kidneys showed decreased p-STAT3 levels in tissue sections (D) (scale bar = 50 μm), as well as in protein lysates (E) after Py treatment. DMSO treatments were used as vehicle controls at P1. (F) qRT-PCR assays showed decreased expression of STAT3 downstream genes in P1 Sclt1^–/– kidneys treated with pyrimethamine. Expression changes are normalized to actin as a housekeeping gene (n = 3; *P < 0.05; error bars: standard deviation).

Pyrimethamine treatment dramatically decreased the amount of p-STAT3 (Fig. 7D and E) and reduced the expression of pro-fibrosis factors (Ctgf and Pai1) and inflammation markers (Il1α, Ilβ and Il6) in the kidneys of Sclt1^–/– mice (Fig. 7F). It reduced other p-STAT3 downstream targets (such as Hif1α and Vegf) in WT and Sclt1^–/– mice to a similar extent (Fig. 7F). Pyrimethamine had no noticeable effect on the morphology and histology of WT control kidneys. These experiments support the concept that anti-STAT3 treatment using pyrimethamine can effectively correct the cystic kidney phenotype of Sclt1^–/– mice through suppression of proliferation, inflammation, and fibrosis.

Discussion

We have identified a novel mouse line with an insertional mutation in the Sclt1 gene. As a centriole distal appendage component, SCLT1 is necessary for ciliogenesis (23). In line with this, we demonstrated that the inactivation of Sclt1 results in a mutant phenotype resembling other ciliopathies. Mutations in the Sclt1 gene have recently been identified in OFD syndrome type IX, a rare human inherited disease (24). A primary myelofibrosis (PMF) patient has been shown to carry a deletion between introns 2 and 3, resulting in a truncated Sclt1 transcript missing the 5′ end and exons 1-2 (47). Sclt1 has also been reported as one of the major driver oncogenes in breast cancer, with mutation prevalence over 5% (48). The lack of animal models with Sclt1 mutations has limited our understanding in the pathomechanisms of these diseases and hindered the development of therapeutic strategies.

Here, we show that Sclt1 loss in mice leads to preaxial polydactyly, cleft palate, short gut, and rapidly progressing cystic kidney disease (see Fig. 1). These phenotypes overlap with OFD syndrome manifestations, although the ciliopathic phenotypes are more severe, with a higher penetrance in our KO model than in the OFD patients. This may be because the mutations in the SCLT1 gene of OFD Type IX cases are hypomorphic or neomorphic. Primary myelofibrosis is a myeloproliferative neoplasm associated with bone marrow fibrosis, splenomegaly, and extramedullary hematopoiesis (49,50). Approximately 50–60% of PMF patients have a JAK2^V617F mutation, and the first FDA-approved oral JAK1/2 inhibitor (ruxolitinib) can significantly reduce splenomegaly and can ameliorate debilitating myelofibrosis-related symptoms (51,52). Because of the high death rate of Sclt1^–/– mice postnatally, we were unable to assess whether they develop a typical PMF phenotype, which is most often diagnosed in persons over the age of 60 (53). A conditional Sclt1 allele that circumvents the early lethal phenotype may be suitable for modeling PMF in mice. Sclt1^–/– mice showed a distinctive intestinal shortening. This phenotype has not been reported in OFD and PMF patients, so its clinical relevance to OFD and PMF needs further evaluation. In addition, whether SCLT1 regulates gut length through mechanisms similar to those regulating kidney development also need to be determined.

The pathogenesis of renal cysts involves multiple cellular abnormities, including increased cell proliferation, fluid secretion, apoptosis, de-differentiation, altered polarity and fibrosis at different phases during PKD progression (12,54). The abnormal proliferation in tubular epithelial cells was crucial for both the initiation and growth of kidney cysts (55). We found that Sclt1 loss led to increased Ki67 expression in renal cells and dramatically elevated PKA, ERK1/2 and STAT3 signaling, similar to changes in cystic kidneys caused by PKD1 or PKD2 mutations (56). Our study also revealed that SCLT1 interacted with PC2 and that overexpressed PC2 could ameliorate PKA activation in Sclt1^KD HEK293 cells (see Fig. 3). These results support the concept that Sclt1 loss may disrupt PC2 function and cause excessive PKA signaling, which can activate the ERK and STAT3 pathways to promote renal cell hyperproliferation. How the SCLT1-PC2 interaction modulates PC2 signal transduction still needs to be clarified.

Similar to other cystic kidney mouse models, the kidneys in the Sclt1^–/– model showed markedly enhanced fibrosis. Pro-fibrotic TGF-β signaling was dramatically increased in parallel with cystic kidney progression, but signaling was unchanged in the newborn Sclt1^–/– kidney (data not shown), suggesting that cystic kidney onset is not directly mediated by TGF-β signaling. In addition, it was reported that Ift88 mutant (Tg737) mice had decreased TGF-β signaling (57). Taken together, the increased TGF-β signaling in Sclt1^–/– and other cystic kidney mouse models is likely because of a net effect of disease progression. For example, chronic inflammation in PKD may escalate TGF-β signaling to advance fibrosis, and/or cells with high TGF-β signaling may become enriched in the later stages of cystic kidney diseases.

STAT3 signaling is a key player in kidney cystogenesis, promoting cell proliferation and fibrosis, and the STAT3 inhibitor pyrimethamine has been used to treat Pkd1 KO mice (46,58). Our study showed that pyrimethamine could also partially correct the cystic kidneys of Sclt1^–/– mice. This result indicated that STAT3 inhibition may be useful as a treatment for SCLT1-related diseases such as OFD and PMF. More broadly, it may be useful as a general therapy for ciliogenesis-defect-related cystic kidney diseases. However, pyrimethamine was also found to be competitive inhibitor of dihydrofolate reductase (DHFR) and cytochromes P450 (CYP) (59,60). DHFR is a key enzyme in the redox cycle for the production of tetrahydrofolate, a cofactor that is required for the synthesis of DNA and proteins; the inhibition of DHFR can limit the growth and proliferation of cells (61,62). Cytochromes P450 are a group of heme-thiolate monooxygenases whose metabolites regulate renal tubular and vascular function and renal epithelial cell proliferation (63–65). Thus, the beneficial outcome of pyrimethamine in the cystic kidney may reflect the combined inhibition of STAT3, DHFR, and CYP. In the future, further treatments using specific inhibitors or genetic approaches are needed to define the contribution of STAT3 as a driver of Sclt1-loss-induced renal cyst growth.

Material and Methods

Animals

Sclt1^–/– mice were generated by intercrossing the transgenic mouse line CAGGS-SB10, a kind gift from Dr David Largaespada (University of Minnesota) (26). All mice were maintained and used following the approval of the Institutional Animal Care and Use Committee (IACUC) at Van Andel Research Institute and performed at a facility accredited by the American Association for Accreditation of Laboratory Animal Care (AALAC).

Antibodies

Anti-acetylated tubulin (IF: 1/200; Sigma, #T6793), anti-FLAG (WB: 1/1000, IP: 1/50; Sigma, #F1840), anti-ERK1/2 (WB: 1/1000, IHC: 1/200; Cell Signaling Technology, #4695), anti-p-ERK1/2 (WB: 1/1000, IHC: 1/200; Cell Signaling Technology, #4370), anti-cleaved-caspase3 (WB: 1/1000, IHC: 1/200; Cell Signaling Technology, #9664), anti-p-SMAD2/3 (WB: 1/1000, IHC: 1/200; Cell Signaling Technology, #8828), anti-p-SMAD1/5/8 (WB: 1/1000; Cell Signaling Technology, #9511), anti-p-STAT3 (Tyr 705) (WB: 1/1000, IHC: 1/200; Cell Signaling Technology, #9145), anti-p-CREB (IHC: 1/200; Cell Signaling Technology, #9198), anti-V5 (WB: 1/1000, IP: 1/50; Cell Signaling Technology, #13202) and anti-Ki67 (IHC:1/500; Pirece, #MA5-14520).

Plasmids, transfection and luciferase assay

Sclt1 cDNA was cloned into the expression vector pcDNA3.1-V5-his (Invitrogen). Full-length PKD2 cDNA was cloned into the expression vector p3X Flag-CMV-10. Luciferase vectors: pLentiEKAR2G2 (ERK activity reporter), pJC6-GL3 (c-Jun promoter), CRE/CREB-luci (cAMP/CREB activity reporter) SBE-luci (TGF-β signaling reporter) and ID1-luci (BMP signaling reporter) were all purchased from Addgene. Scrambled shRNA and SCLT1 shRNA (NM_144643.2-858s21c1, Sigma Aldrich) plasmids were co-expressed with pMD2.G and psPAX2 using X-Fact transfection reagent (Clontech) for the production of shRNA virus from HEK293 cells. We then generated SCLT1^KD and control HEK293 cells after selection using 2 μg/ml puromycin, according to the protocol of the manufacturer.

The luciferase assays were performed as described previously (66). Briefly, SCLT1^KD and control HEK293 cells were plated on 24-well plates and transfected with a combination of plasmids using X-Fact transfection reagent (Clontech). The renilla luciferase plasmid was used to normalize transfection efficiency. The dual luciferase assay was conducted using a kit from Promega and quantified on the SYNERGY NEO microplate reader (BioTek)

Fluorescent in situ hybridization (FISH)

Fluorescent in situ hybridization followed protocols described previously (67,68). Briefly, mouse chromosomes were prepared from early passage lung fibroblasts harvested from a homozygous male mouse. The Sleeping Beauty transposase probe was labeled with digoxigenin (Boeringer Mannheim) by nick translation. A solution containing 200 ng of labeled DNA, 10 mg of salmon sperm DNA and 5 mg of mouse Cot-1 DNA (Gibco BRL, for reducing background signals) was used to probe previously G-banded slides. Slides were hybridized overnight and washed with 1× saline-sodium citrate (SSC) buffer at 72 °C. Probe DNA was detected with anti-digoxigenin FITC (Boehringer Mannheim) and chromosomes were counterstained with 0.2 mg/ml propidium iodide in an antifade solution. Images were captured using a PowerGene probe analysis system.

Histology and immunohistochemistry/immunofluorescence

Mouse tissues were fixed in 4% paraformaldehyde, dehydrated and paraffin-embedded, and sectioned at 5-µm thickness. Staining with hematoxylin and eosin (H&E) followed normal procedures. For immunohistochemistry, deparaffined and rehydrated sections were incubated in an antigen unmasking solution (10 mM sodium citrate buffer, pH 6.0) at 95 °C for 15 min, then quenched in 5% H₂O₂ for 10 min. After rinsing in PBS, the sections were blocked in the normal serum blocker (1% normal serum and 1% BSA in PBS) for 1 h, then incubated with primary antibodies overnight at 4 °C. After washing three times in PBS, sections were incubated with biotinylated secondary antibody (1/400, Vector Laboratories) for 2 h at room temperature, then incubated with avidin–biotin–peroxidase complex (ABC) reagent for 30 min at room temperature, and detected by peroxidase substratereaction (Vector Laboratories). Sections were counter-stained with hematoxylin and mounted.

For immunofluorescence, the rehydrated and unmasked sections were blocked in 5% BSA-PBS for 30 min and then incubated with anti-acetylated tubulin for cilia detection or anti-γ-tubulin for centrosome detection. After detection with florescence-labeled secondary antibody, the sections were counter-stained with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories) and mounted.

For DBA and LTL staining, the kidney sections were deparaffinized and rehydrated, then incubated in a mix of 10 µg/mL each of fluorescein–Lotus tetragonolobus lectin (LTL) and rhodamine–Dolichos biflorus agglutinin (DBA, Vector Laboratories) for 2 h at room temperature. Slides were rinsed in PBS, counterstained with DAPI, and mounted.

Blood urea nitrogen (BUN) measurement

Mouse serums were separated from blood collected from the P1 mice. Serum BUN was measured by a standard autoanalyser technique using a VetScan Chemistry Analyzer (Abaxis, Inc., Union City, CA). The serum of P1 mice was diluted 1:5 with saline prior to the measurement (69).

Western blot and co-immunoprecipitation

Total protein was extracted from kidney tissue using a radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor (cocktail and PMSF) and quantified using a BCA assay (Pierce). Equal amounts of protein were loaded onto an SDS-PAGE gel and subjected to standard western blot procedures.

The co-immunoprecipitation (Co-IP) assays were conducted following protocols described previously (66). Briefly, cells were lysed in the IP buffer (50 mM Tris-HCl [pH 7.4], 0.1% Triton X-100, 150 mM NaCl, 10 nM N-ethylmaleimide [Sigma], plus 1:100 protease inhibitor cocktail [Roche]). The cell lysates were incubated with anti-FLAG, anti-V5 antibody or normal IgG control (all at 1 µg/ml) overnight at 4 °C and subsequently precipitated with Protein G magnetic beads (Bio-Rad) for 4 h. The beads were washed with IP buffer for 3 times, boiled in Laemmli sample buffer, and subjected to western blotting.

Quantitative RT-PCR (qRT-PCR) and RNA-seq analyses

Total RNA was extracted from whole kidney using TRIzol (Invitrogen) using the protocols described previously (70), by use of a GenElute Mammalian Total RNA kit with on-column DNase digestion (Sigma). First-strand cDNA was generated from 1 μg RNA using SuperScript VILO cDNA Synthesis Kit (Invitrogen). Real-time PCR was performed on a StepOnePlus system (Company) using Fast SYBR Green Master Mix (Invitrogen). The relative gene expression levels were calculated using 2^–ΔΔCT method. The β-actin gene was used as an internal expression control. The specific primer sequences used in the study are provided in Supplementary Material, Table S1.

Purified RNA from kidney samples was sequenced using the service provided by Beijing Genomics Institute (BGI), and analyzed using the Gene Set Enrichment Analysis (GSEA) software developed by Broad Institute. Differentially expressed genes were ranked based on the approaches described in (71).

Cyst index measurement

The cyst index was quantified in the H&E stained whole-kidney sections using Image Pro Plus v5 software (Media Cybernetics) with the formula: Cyst index (%) = (cystic area/total kidney area) × 100. Two sections from both kidneys were analyzed for each mouse, and the averaged numbers were used for final statistical analysis.

Administration of pyrimethamine

Pyrimethamine (Sigma-Aldrich) dissolved in 100% dimethyl sulfoxide (DMSO) was administered to E12.5 embryos through daily gavage to pregnant female mice for 7 d (30 mg/kg of body weight) (46). The same volume of DMSO was use as a vehicle control.

Statistics

All statistical results are presented as the mean ± standard deviation (S.D.). The differences between two groups were calculated using the two-tailed Student’s t-test. P values less than 0.05 were considered statistically significant.

Supplementary Material

Supplementary Material is available at HMG online.

Acknowledgements

We thank Dr David Largaespada (University of Minnesota) for providing the CAGGS-SB10 transgenic mice, members of the Yang laboratory and other members of the Program for Skeletal Disease and Tumor Metastasis at Van Andel Research Institute for discussion, the Vivarium and Transgenics Core for animal technical assistance, Ms Megan Bowman for assistance with resembling the mutant Sclt1 mRNA from the RNA-seq data, Mr David Nadziejka for assistance with technical editing of this manuscript.

Conflict of Interest statement. None declared.

Funding

This work was supported by VARI startup funds (T.Y) and partially supported by the Chinese 111 Project Grants B06018 and B16036 (L.Z).