Background
Schimke immuno-osseous dysplasia (SIOD, OMIM 242900) is an autosomal recessive disease; its prominent features are facial dysmorphism, hyperpigmented macules, focal segmental glomerulosclerosis (FSGS), spondyloepiphyseal dysplasia, and T-cell immunodeficiency [
1‐
3]. Additional features include hypothyroidism, abnormal dentition, bone marrow failure, thin hair, corneal opacities, arteriosclerosis, cerebral ischemia, and migraine-like headaches [
2‐
5].
The renal disease begins as proteinuria, progresses to steroid-resistant nephropathy, and ultimately advances to end-stage renal disease [
4,
6]. FSGS is the predominant renal pathology and is refractory to treatment with glucocorticoids, cyclosporine A, and cyclophosphamide [
4,
6]. Suggesting a cell autonomous mechanism for the renal disease, renal transplantation is efficacious, and the disease does not recur in the graft [
2,
4,
5].
Biallelic mutations of the
SWI/SNF-related
matrix-associated
actin-dependent
regulator of
chromatin, subfamily
A-
like
1 (
SMARCAL1) gene cause SIOD [
7].
SMARCAL1 encodes a DNA annealing helicase that is a distant member of the SWI/SNF family of ATP-dependent chromatin remodeling proteins [
8]. SMARCAL1 recognizes DNA structure, binds to open chromatin, is involved in the DNA damage response [
9,
10] and DNA replication fork restart [
11,
12], and, along with genetic and environmental factors, alters gene expression [
13].
Gene expression changes appear critical to SIOD pathology. Full or partial explanations for the vascular disease and T-cell immunodeficiency of SIOD patients are respectively decreased expression of elastin (
ELN) in the aorta [
14‐
16] and of
inter
leukin
7 receptor alpha chain (
IL7R) in the T cells [
17‐
19].
Based on these findings, we hypothesized that SMARCAL1 deficiency causes the renal disease of SIOD by altering gene expression. Studies of other glomerulopathies find increased Wnt [
20‐
23] and Notch signaling [
24‐
27] as causes of podocyte dysfunction. Canonical Wnt pathway activation proceeds via inhibition of β-catenin ubiquitination, saturation of the β-catenin destruction complex, cytoplasmic accumulation and nuclear translocation of newly synthesized unphosphorylated β-catenin, and subsequent activation of target gene transcription through interaction with transcription factors and transcriptional co-activators [
28]. Notch pathway activation involves proteolytic cleavage of the Notch transmembrane receptor by an ADAM metalloproteinase and the γ-secretase complex, nuclear translocation of the released Notch1 intracellular domain (NICD), and subsequent activation of target gene transcription through interaction of the NICD with transcription factors and transcriptional co-activators [
29]. Wnt and Notch signaling are critical for kidney development and become undetectable in the glomeruli of the postnatal kidney [
26,
30].
Analyses presented herein showed upregulation of the Wnt and Notch signaling pathways in the SIOD kidney and genetic interaction between the Drosophila SMARCAL1 homologue and genes encoding components of the Wnt and Notch pathways. We suggest therefore that the upregulation of the Wnt and/or Notch pathways contributes to the renal disease in SIOD.
Methods
Patients and human tissues
The guardians of the patients referred to this study signed informed consent approved by the Research Ethics Board of the University of British Columbia (Vancouver, BC, Canada). Autopsy and biopsy tissues were obtained according to the protocol approved by the University of British Columbia (Vancouver, BC, Canada). The renal parameters and the
SMARCAL1 mutations of the SIOD patients included in the study are listed in Table
1 and Additional file
1: Table S1, respectively.
Table 1
The renal parameters of the SIOD patients included in this study
SD4b | 3 | 8 | + | + | + | ? | − | n/a | − | n/a | FSGS |
SD26 | <4 | 8 | + | + | + | + | + | 5 | − | n/a | FSGS |
SD60 | 7 | 13.7 | + | + | + | ? | + | 12.5 | + | 13 | FSGS |
SD79 | <4 | 10 | + | − | + | − | − | n/a | − | n/a | FSGS |
SD120 | 4.5 | 5.4 | + | + | + | + | − | n/a | − | n/a | FSGS |
SD121 | 2.5 | 4.8 | + | − | + | + | − | n/a | − | n/a | Diffuse podocytopathy with early features of FSGS |
SD131 | 3 | 4.6 | + | + | + | + | + | 3.8 | − | n/a | Global glomerulosclerosis likely secondary to FSGS |
SD146 | 2 | 4 | − | − | + | + | − | n/a | − | n/a | FSGS |
In accordance with institutional policies as approved by the Institutional Review Board (41557) at the University of Washington, human fetal kidney from second trimester elective terminations were provided as de-identified specimens by the Laboratory of Developmental Biology at the University of Washington (Seattle, WA), a National Institute of Child Health & Human Development supported program. De-identified control specimens provided according to the protocol H06-70283 approved by the Clinical Research Ethics Board at the University of British Columbia (Vancouver, BC, Canada) included renal biopsy sections from ten pediatric patients with isolated FSGS, postmortem kidney tissue from four pediatric patients, a skin biopsy from a 16-year-old female, and adenoma tissue from a 17-year-old female with familial adenomatous polyposis. Sample characteristics and use are summarized in Additional file
1: Table S2.
Drosophila melanogaster lines
The loss-of-function mutant
Marcal1
del
and the
Marcal1 overexpression transgenic line
pUAST-Marcal1/CyO;
tubulin-GAL4/TM3, Sb
1
have been previously described [
13] (Additional file
1: Figure S1). The
C96-GAL4 UAS-Hrs/MKRS transgenic line, used to control for non-specific interactions with the GAL4-UAS system, was a gift from Dr. Hugo Bellen (Baylor College of Medicine, Houston, TX, USA). All other
Drosophila stocks were obtained from the Bloomington
Drosophila Stock Center (Bloomington, IN, USA).
Total RNA was extracted from flash frozen kidney pulverized with a Bessman tissue pulverizer (Spectrum Laboratories, Rancho Dominguez, CA, USA) or from 8 Drosophila adult female flies of each genotype by using the RNeasy Mini Kit (Qiagen, Toronto, ON, Canada). Total RNA from formalin-fixed paraffin-embedded (FFPE) fetal kidney was isolated using the RNeasy FFPE Kit (Qiagen, Toronto, ON, Canada). Genomic DNA was removed by on-column DNase I digestion (Qiagen, Toronto, ON, Canada).
RNA-seq and KEGG pathway analysis
Strand-specific, paired-end RNA-seq on poly(A) RNA was performed by Macrogen (Seoul, Korea) using the TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA) and the HiSeq 2000 System (Illumina, San Diego, CA). This kit depleted the ribosomal RNA (rRNA) using Ribo-Zero rRNA reduction chemistry. Quantification was performed by calculating fragments per kilobase per million mapped reads (FPKM). Prior to fold change calculation and log
2 transformation, a pseudocount of 1 was added to each FPKM value to reduce the inherent bias of finding gene expression changes in those genes where one sample has very little or no detectable gene expression [
31]. The threshold for differential gene expression between the kidney from the SIOD patient and sex-matched unaffected control was set at log
2 fold change (i.e., log
2 (FPKM
SIOD + 1/FPKM
UNAFFECTED + 1)) > 1 or < −1. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed with the online bioinformatic resource
Database for
Annotation,
Visualization, and
Integrated
Discovery (DAVID) version 6.7 available at
https://david.ncifcrf.gov.
Reverse transcription
For total RNA extracted from flash frozen kidney, reverse transcription was performed with the RT2 First Strand Kit (Qiagen, Toronto, ON, Canada). For total RNA extracted from FFPE kidney or adult flies, reverse transcription was performed with the qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, USA).
Gene expression arrays
The Wnt (PAHS-043Y) and Notch (PAHS-059Y) Signaling Pathway Plus PCR Arrays (Qiagen, Toronto, ON, Canada) and the RT2 Real-Time SYBR Green/Rox PCR Master Mix (Qiagen, Toronto, ON, Canada) were used to assess mRNA levels between the sex-matched unaffected control and the SIOD kidney according to the manufacturer’s specifications. The threshold for calling differential mRNA levels was a log2 fold change > 1 or < −1 and a p value of less than 0.05.
Quantitative PCR
SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Mississauga, ON, Canada) was used with the StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) for quantitative PCR. Human
GAPDH and
Drosophila Gapdh2 housekeeping genes were used as endogenous controls. The primer sequences used in this study are listed in Additional file
1: Table S3.
Indirect immunofluorescence
FFPE sections of tissue or cell pellets were cut at 5 microns. Following deparaffinization and rehydration, heat induced epitope retrieval was performed with sodium citrate buffer (10 mM sodium citrate, 0.05 % Tween 20, pH 6.0). Endogenous peroxidases were inactivated for 1 h at room temperature by incubating the sections with peroxidase quenching buffer (3 % hydrogen peroxide in 1× phosphate-buffered saline (PBS), 0.1 % Tween 20, pH 7.4 (PBSTw) for unphosphorylated β-catenin immunofluorescent staining or 1× PBS, 0.2 % Triton X-100, pH 7.4 (PBST) for the Notch1 intracellular domain (NICD) immunofluorescent staining). Non-specific protein binding was blocked by incubating the sections with blocking buffer (20 % normal goat serum, 10 % bovine serum albumin, 1× casein (Vector Laboratories, Burlington, ON, Canada) in PBSTw or PBST) overnight at 4 °C. Endogenous biotin, biotin receptors, and avidin binding sites were blocked with the Avidin/Biotin Blocking Kit (Vector Laboratories, Burlington, ON, Canada).
Rabbit anti-unphosphorylated β-catenin (clone D13A1, Cell Signaling Technology, Danvers, MA, USA) or rabbit anti-NICD (ab8925, Abcam, Toronto, ON, Canada) were used as primary antibodies. A biotinylated anti-rabbit IgG secondary antibody was used to detect the primary antibodies. Horseradish peroxidase-conjugated streptavidin was then used to detect the biotinylated anti-rabbit IgG secondary antibody. Subsequently, tyramide labeling was performed using Alexa Fluor 594 tyramide (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). ProLong Gold Antifade Mountant with 4′, 6-diamidino-2-phenylindole (DAPI) (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was used to mount the sections and counterstain the DNA. Representative images were acquired using a 20×/0.75 Plan-APOCHROMAT, 40×/1.3 oil DIC Plan-NEOFLUAR, or 100×/1.30 oil Plan-NEOFLUAR objective lens on an Axiovert 200 inverted microscope, an AxioCam MR microscope camera, and the AxioVision software version 4.8 (Carl Zeiss, Toronto, ON, Canada). The glomerular β-catenin signal was quantified for each sample (see Additional file
1: Methods for further details).
Drosophila genetics studies
We performed an overexpression and loss-of-function genetic screen in
Drosophila to determine whether the
SMARCAL1 homologue
Marcal1 genetically interacts with Wnt and Notch pathway genes (see Additional file
1: Methods for further details).
Statistics
For the KEGG pathway analysis, enrichment p values were corrected for multiple comparisons by the Bonferroni method. A p value of less than 0.05 was considered statistically significant. For the PCR expression arrays, data were analyzed by the 2-tailed Student’s t-test. A p value of less than 0.05 was considered statistically significant.
Discussion
Herein we identify increased signaling of the Wnt and Notch pathways as potential causes for the renal disease in SIOD. Most SIOD kidneys exhibited increased levels of unphosphorylated β-catenin and NICD respectively indicating increased Wnt and Notch pathway activity. Similarly, most isolated FSGS kidneys had upregulated unphosphorylated β-catenin and NICD. The failure to observe increased unphosphorylated β-catenin and NICD in the renal graft of an SIOD patient suggests that these molecular findings are inherent to the diseased kidney and not induced from outside of the kidney. The genetic interaction between Marcal1 and the Wnt and Notch pathway genes in Drosophila suggests that that the altered signaling of these pathways is a direct or indirect consequence of SMARCAL1 deficiency.
The consistency of increased markers for both activation of the Wnt and Notch pathways in both SIOD and isolated FSGS control kidneys suggests that activation of both pathways underlie the renal disease of SIOD and isolated FSGS (Fig.
4c). Activation of both pathways is not essential for induction of SIOD renal disease or isolated FSGS, however, because a few samples showed activation of only one or neither of these pathways (Fig.
4c).
Based on our observations in the 15-week-gestation fetal kidney, the potentially pathological activation of Wnt and Notch signaling in the SIOD kidneys appears to arise after this stage of renal development. Further studies are required to define precisely the timing of the pathological activation of these pathways.
Although the Notch pathway gene expression changes were not identified in the KEGG pathway analysis of the transcriptome, the high level of crosstalk between the Wnt and Notch signaling pathways [
33], and their role in kidney development and disease prompted us to also investigate the upregulation of the Notch pathway as a potential cause for the FSGS in SIOD. Possible reasons for the transcriptome analysis not detecting the upregulation of the Notch pathway include pathway size bias inherent to KEGG pathway analysis (the Wnt signaling pathway includes 141 genes, whereas the Notch signaling pathway includes 48 genes) and tissue heterogeneity.
The mechanism by which SMARCAL1 deficiency gives rise to tissue-specific changes in gene expression is incompletely understood. It could arise from a direct consequence of SMARCAL1 deficiency on the DNA structure of a gene or of the genes encoding the transcriptional regulators of that gene. Consistent with this, we previously observed that SMARCAL1 homologues bind transcriptionally active chromatin and modulate gene expression [
13]. Sharma et al. (2015) recently showed that the bovine orthologue of SMARCAL1 negatively and directly regulates the transcription of
MYC by altering the conformation of its promoter [
34]. Alternatively, because stalled replication forks induce epigenetic changes that alter gene expression [
35,
36], impedance of DNA replication fork restart by SMARCAL1 deficiency might contribute to the changes in gene expression. Consistent with the latter possibility, we recently observed hypermethylation of the
IL7R promoter in the T cells of SIOD patients [
19]; reduced
IL7R expression in human CD8
+ T cells is associated with hypermethylation of the
IL7R promoter [
37].
A limitation of the study was the use of whole kidney to profile differential gene expression in an SIOD kidney. Given that the primary lesion is limited to the glomeruli, the affected tissue represents a small fraction of the total tissue. Although several human gene expression studies on FSGS have used isolated glomeruli [
38,
39], others have successfully used renal biopsies [
40]. Similar to other human gene expression studies of FSGS [
38‐
40], the expression of podocyte-specific genes including
NPHS1,
NPHS2, and
WT1 were downregulated in the SIOD kidney, and most of the KEGG pathways that were enriched in our list of upregulated genes were also enriched in the prior studies, including the Wnt signaling pathway [
38].
A second limitation of the study was that only unphosphorylated β-catenin and nuclear NICD were examined by immunofluorescence as measures of pathway activation. This constraint arose secondary to limited tissue. We selected these proteins because they are the primary effectors of and activation markers for the canonical Wnt and Notch signaling pathways. However, Wnt signaling has canonical and non-canonical pathways, and there is also Wnt-independent β-catenin activation [
41]. Notch signaling also has canonical and non-canonical pathways as well as three Notch receptors in addition to Notch1 [
42]. Our findings nonetheless set a precedent for future studies examining the pathogenesis of renal disease in SIOD.
Conclusions
In summary, our findings show that the Wnt and Notch pathways are upregulated in the SIOD patient kidney and that Marcal1, the Drosophila SMARCAL1 homologue, genetically interacts with Wnt and Notch pathway genes. Based on these findings, the renal disease of SIOD is yet another clinically distinctive feature of SIOD likely arising through alterations of gene expression.
Acknowledgements
We are grateful to all of the patients and family members who have contributed to this study. The authors thank Theresa Sturby (Children’s and Women’s Health Centre of British Columbia) for her technical expertise, and Drs. Darren Bridgewater and Alireza Barandaran-Heravi for critical review of this manuscript.
Authors’ contributions
All authors have made substantial contributions to the article by participating in the conception and design (MM, CFB), acquisition of data (MM, CM, KB, KC, YA, AB, DB, MB, JC, EC, AD, GD, M Gentile, M Giordano, AKG, RG, MJ, KK, E Lerut, E Levtchenko, LM, CM, BN, DP, JS, PS, UY, ZY, JZ, GH, CFB), analysis and interpretation of data (MM, CM, KB, KC, BN, CFB), drafting the manuscript (MM, CFB), or revising it critically for important intellectual content (MM, CM, KB, KC, YA, AB, DB, MB, JC, EC, AD, GD, MGentile, MGiordano, AKG, RG, MJ, KK, ELerut, ELevtchenko, LM, CM, BN, DP, JS, PS, UY, ZY, JZ, GH, CFB). All authors read and approved the final manuscript.