Keywords
CAKUT, vitamin A, vitamin D, nutrients, environmental factor, renal development
This article is included in the Cell & Molecular Biology gateway.
CAKUT, vitamin A, vitamin D, nutrients, environmental factor, renal development
In line with the comments from the reviewers, we made the following new analyses and changes:
In the previous version of the manuscript, we presented the overlap analysis of vitamin A and vitamin D target genes with 13 CAKUT-related gene sets. In this revised version, we added a set of CAKUT differentially expressed genes to the analyses. We hence repeated the hypergeometric test for 14 gene sets. Due to the multiple testing correction, previous adjusted p-values slightly changed.
We added explanations for the statistical test, the null hypothesis, and the background sets used in the analyses.
As a supplementary analysis, we calculated the overlap statistics with randomized sampling (in addition to the hypergeometric test in the main manuscript).
As a supplementary file, we provide the overlap of CAKUT causal genes with other CAKUT-related gene sets without considering being a vitamin target, for the interested readers.
We added descriptions for the roles of BMP4, HNF1B, RET and NRIP1, which are CAKUT causal genes and vitamin A targets according to both CTD and Balmer and Blomhoff (2002).
See the authors' detailed response to the review by Olivia Angelin-Bonnet
See the authors' detailed response to the review by Elena Menegola
See the authors' detailed response to the review by Sean Eddy
See the authors' detailed response to the review by Matias Simons
Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) are a group of abnormalities affecting the kidneys and their outflow tracts, including the ureters, the bladder, and the urethra. In the European Union, the overall prevalence of CAKUT (in live plus stillbirths) between 2011 and 2018 was approximately 35:10,000.1 The main anomalies observed in CAKUT are hydronephrosis (13.02:10,000) and multicystic renal dysplasia (4.33:10,000); these are followed by posterior urethral valves (1.26:10,000), bilateral renal agenesis (1.27:10,000) and bladder exstrophy/epispadia (0.62:10,000); many other anomalies with low prevalence can also be observed.1 This clinical variability observed in patients is accompanied by a variable severity of the phenotypes.2
Approximately 40 different genes are known to be associated with monogenic causes of CAKUT in humans, but they explain only 5% to 20% of the cases.3,4 The clinical variability and the complex aetiology of CAKUT cases suggest a multifactorial origin with complex interactions of both genetic and environmental factors contributing to the disease.2,3,5
The role of different environmental factors in CAKUT pathogenesis has been studied previously. It has been shown, for instance, that the drugs inhibiting the angiotensin-converting enzymes cause a specific form of CAKUT, namely tubular dysgenesis.6 Many prenatal and maternal factors were also assessed for their involvement in CAKUT and related anomalies. For example, studies have observed CAKUT associations with pregestational maternal diabetes mellitus,7,8,9 gestational maternal diabetes mellitus,8,10,11 abnormal volume of amniotic fluid,7,10 urogenital infections before the pregnancy,12 any infection during the pregnancy,12 maternal overweight and obesity,7,8,11,13 and lower infant birth weight.7,8,9,10 These maternal and prenatal factors are modulated by environmental risk factors. In particular, maternal diabetes and overweight/obesity as well as low birth weight are related to the nutrition of the maternal-fetal dyad. In this regard, trace nutrients, like vitamins, are likely to play important roles that still await full characterization.14
Vitamins are known to regulate renal development.15,16,17,18 The association of maternal vitamin A deficiency with nephron reduction was studied on a rat model.19 The inactivation of the retinoic acid nuclear receptors also resulted in renal malformations in mice.20 An additional study on rats showed that vitamin A deficiency downregulates RET expression which is essential for epithelial-mesenchymal interaction during renal development.21 Goodyear et al. compared a group of pregnant women in Bangalore with a high (55%) prevalence of vitamin A deficiency with a group of pregnant women in Montreal with negligible vitamin A deficiency.22 The authors found that renal volume in newborns, adjusted for body surface area, was significantly lower in the vitamin A-deficient population. While the two populations are different for other genetic and environmental reasons, these results suggest that vitamin A deficiency may have a role in human CAKUT. El-Khashab et al. reported a similar finding in their study on a cohort of Egyptian mother-child pairs;23 children of vitamin A deficient mothers had significantly lower kidney sizes.
The research on the effects of vitamin D deficiency on kidney development has generated interesting results. Rogers et al.24 observed that incubation of metanephroi with vitamin D3 prior to implantation into adult rats increased the number of glomeruli. Conversely, Maka et al.25 and Nascimento et al.26 observed that maternal vitamin D deficiency stimulated nephrogenesis in rats. Following these studies, Boyce et al. examined the long term effects of maternal vitamin D deficiency and observed that adult male rat offspring of vitamin D deficient dams had reduced creatinine clearance, indicating reduced renal functional capacity.27 They also observed a significant upregulation of renal renin mRNA expression in fetuses and adult male offspring, and reported smaller kidneys in the offspring. Miliku et al. examined the association of 25-hydroxyvitamin D levels during mid-pregnancy with childhood kidney outcomes among 4212 mother-child pairs.28 They observed that children of mothers who were vitamin D deficient during pregnancy had a larger combined kidney volume compared to children of mothers who had optimal vitamin D levels.
In this study, we examined whether vitamin A and vitamin D target genes are related to gene sets involved in CAKUT and renal system development. Our aim is to generate hypotheses for the involvement of these vitamins in the disease aetiology. We first created a list of vitamin A and vitamin D target genes, extracting information from the Comparative Toxicogenomics Database (CTD)29 as well as from two publications.30,31 We then constructed different gene sets relevant to CAKUT, including genes mutated in monogenic forms of CAKUT, genes involved in renal system development, and genes involved in different pathways of interest. Finally, we performed overlap analyses to identify the genes involved in these different gene sets as well as the significant overlaps.
We first queried vitamin A and downloaded all gene interactions of vitamin A and its descendants from the Comparative Toxicogenomics Database (CTD).29 We selected only the interactions supported by at least two references. This gave a list of 1086 target genes.
As an independent source, we used the data from the study of Balmer and Blomhoff.30 In this study, the authors reviewed published data from 1191 articles to identify genes regulated by retinoic acid (the active metabolite of vitamin A) in humans and other species. Only a small subset of these articles are part of the CTD curation. The authors provide a list of retinoic acid target genes split into four categories, ranging from strong evidence to indirect regulation through a transcriptional intermediary. We selected the genes from all of these categories, converted gene symbols to human orthologs, and updated gene symbols when necessary using the HUGO Gene Nomenclature Committee (HGNC), Rat Genome Database (RGD), Mouse Genome Database (MGD), BioMart and SynGo. The final list of genes obtained from Balmer and Blomhoff contains 521 target genes, of which 229 are common with genes from CTD.
We queried vitamin D and downloaded all gene interactions of vitamin D and its descendants from the CTD. We selected only the interactions supported by at least two references, and obtained a list of 263 target genes.
Ramagopalan et al.31 identified 230 genes with significant expression changes in response to vitamin D on lymphoblastoid cell lines using microarrays. This publication is not part of the CTD curation, hence it brings additional and independent information. We checked the gene names using HGNC and obtained a list of 210 target genes.
There are only 15 common genes between the list from CTD and the list from Ramagopalan et al. The combined list of vitamin D target genes (458 genes) has 134 genes in common with the combined list of vitamin A target genes (1378 genes).
We collected complementary gene sets relevant to CAKUT and renal system development from several sources, outlined below.
CAKUT causal genes set
First, we created a set of genes known to be mutated in the monogenic form of CAKUT. To do so, we combined two lists of genes provided in the studies of van der Ven et al.3,4 Only six genes are different between these two lists, and their union leads to 42 causal genes (see Extended data32).
CAKUT differentially expressed genes set
Jovanovic et al.33 identified 27 upregulated and 51 downregulated genes as a result of the transcriptome profiling of 15 CAKUT and 7 control ureter samples. We retrieved these differentially expressed genes (DEGs) from the supplementary file they provided. We updated gene names with the help of HUGO Gene Nomenclature Committee (HGNC) and obtained a final list of 74 DEGs (see Extended data32).
Gene Ontology kidney development gene sets
We extracted the genes annotated with the three terms below from Gene Ontology (GO).34,35 Please note that these terms have parent-child relationships.
• GO:0072001 Renal system development (315 genes)
• GO:0001822 Kidney development (306 genes)
• GO:0060993 Kidney morphogenesis (96 genes)
Reactome pathways
We selected different pathways of interest for CAKUT. The renin-angiotensin system is essential for kidney development and mutations in the genes of this system result in CAKUT.36 In addition, Davis et al. discussed the role of RET signaling in kidney development and CAKUT.37 In multiple studies38–43 the roles of WNT, NOTCH, and Hedgehog signaling in kidney development and kidney diseases are discussed. Based on these studies we selected the following Reactome44 pathways:
• R-HSA-2022377 Metabolism of angiotensinogen to angiotensins (18 genes)
• R-HSA-8853659 RET signaling (38 genes)
• R-HSA-195721 Signaling by WNT (328 genes)
• R-HSA-157118 Signaling by NOTCH (233 genes)
• R-HSA-5358351 Signaling by Hedgehog (149 genes)
WikiPathways
From WikiPathways Rare Disease Portal45 we obtained the following four CAKUT-related pathways:
We computed the overlaps between vitamin target gene sets and the CAKUT gene sets of interest defined previously. After obtaining the overlap results, we calculated the significance of the overlaps using the hypergeometric test. Hypergeometric test calculates the statistical significance of getting k successes in n draws without replacement from a finite population of size N that contains a total of K successes. In our context, k is the number of genes overlapping with a specific gene set, K is the size of that gene set, n is the number of vitamin targets, and N is the background gene set (also known as the reference set). We set N to the number of annotated genes in the database from which we obtained the tested gene set. For the CAKUT causal genes set and the differentially expressed genes set, we used the largest N among the different databases, which corresponds to Gene Ontology. The null hypothesis is that the vitamin target genes are not associated with the gene set and the selection of k genes from that gene set is just a result of simple random sampling.
We performed a hypergeometric test for all the gene sets. A consequence of multiple testing is false discoveries in which null hypothesis is rejected erroneously. To control for the false discovery rate, we used the Benjamini-Hochberg correction method (BH adjusted).
We performed two supplementary analyses.
• We calculated the overlap statistics with a randomized sampling approach instead of hypergeometric test as a confirmation
• We calculated the overlaps of the CAKUT causal genes set with the other CAKUT-related gene sets (without considering being a vitamin target)
The details of the supplementary analyses and results are provided in Extended data.32
The results of the overlap analyses between vitamin A and D target genes and CAKUT-related gene sets are presented in Tables 1 and 2, and the corresponding data is available in Underlying data.32
Pub: Publications,3,4,33 GO: Gene Ontology, Reac: Reactome, WP: WikiPathways.
Pub: Publications,3,4,33 GO: Gene Ontology, Reac: Reactome, WP: WikiPathways.
Vitamin A target genes obtained from both CTD and Balmer and Blomhoff are significantly enriched in the set of CAKUT causal genes, with an overlap of ten and six genes, respectively (Table 1). Vitamin A target genes obtained from CTD are also significantly enriched in the set of CAKUT differentially expressed genes (DEGs).
Significant overlaps are observed focusing on GO terms related to renal system development, kidney development, and kidney morphogenesis. These results are expected due to the recognized role of vitamin A in differentiation.46,47 It should be noted that all overlapping CAKUT causal genes except NRIP1 are also part of the kidney development GO term.
Four genes (BMP4, HNF1B, RET, NRIP1) appear as particularly interesting as they are CAKUT causal genes and vitamin A targets according to both CTD and Balmer and Blomhoff. BMP4 is a growth factor of the TGF-beta family involved in a wide variety of developmental processes.48 BMP4 inhibits ectopic budding of the ureteric tips and promotes elongation of the ureter.49 RET is a tyrosine-protein kinase receptor, involved in a large variety of cellular processes. RET signaling is important, in particular, for the terminal growth of the nephric duct, and for initial formation and outgrowth of the ureteric bud.50 The Hepatocyte Nuclear Factor 1-beta (HNF1B) is a transcription factor involved in ureteric bud branching and induction of nephrogenesis. It is required for normal S-shaped bodies patterning and subsequent morphogenesis of all nephron segments.51 Finally, Nuclear receptor interacting protein 1 (NRIP1) modulates the activity of nuclear receptors. It has been shown to play roles in renal malformations via deregulations of retinoic acid signaling.52
Concerning the overlap with Reactome pathways, the vitamin A target genes obtained from CTD are significantly enriched in signaling by NOTCH. The NOTCH signaling pathway is mainly involved in cell-to-cell communication,53 and it plays a role in the development of most organs and tissues.54 Other Reactome terms also have overlapping genes, even if not significant. This includes the WNT signaling pathway (which is involved in developmental processes such as patterning, differentiation/proliferation shift, and migration) and the metabolism of angiotensinogen to angiotensins pathway, from which CTSD, CTSG and MME are vitamin A targets and play roles in the generation of different angiotensin peptides.55,56,57 The impaired conversion of angiotensin I to angiotensin II is the pharmacodynamic adverse mechanism underlying the fetopathy caused by ACE-inhibiting drugs.58 One main feature of this teratogen-induced fetopathy, namely renal tubular dysgenesis, belongs to the CAKUT spectrum. In addition, an overproduction of angiotensin II might have a role in CAKUT anomalies involving renal tubules, as observed in cases of the autosomal recessive polycystic kidney disease.59
Finally, vitamin A target genes extracted from both CTD and Balmer and Blomhoff are significantly enriched in the development of ureteric collection system, genes controlling nephrogenesis, and GDNF/RET signaling axis pathways, as defined in WikiPathways. The target genes extracted from CTD are also significantly enriched in nephrogenesis. Signaling by GDNF through the RET receptor is required for normal growth of the ureteric bud.60 Localized expression of GDNF by the metanephric mesenchyme is important to elicit and correctly position the initial budding event from the Wolffian duct and to promote the continued branching of the ureteric bud.
Overall, the significant overlaps observed between vitamin A and the CAKUT gene sets indicate that vitamin A might be relevant to a broad range of urogenital abnormalities.
In the analyses of vitamin D target gene sets, we overall observed very few significant overlaps with CAKUT-related gene sets. Vitamin D target genes obtained from CTD are only significantly enriched in renal system development and kidney development GO terms. It should be noted that in each case 70% of the enriched genes are also vitamin A target genes (according to CTD or Balmer and Blomhoff).
CTSZ, which cleaves angiotensin I to yield angiotensin II,61 is the only vitamin D target in the metabolism angiotensinogen to angiotensins pathway. Thus, within CAKUT, vitamin D might also have a role in renal tubular defects related to anomalies of the renin-angiotensin system.
WNT and NOTCH signaling pathways have genes that are targets of vitamin D but there is a small, non-significant overlap, and the overlapping genes are not the WNT and NOTCH receptors nor ligands.
In this study we examined the overlaps of vitamin A and vitamin D target gene sets extracted from different databases and publications with CAKUT-related genes and biological processes. While we only observed significant enrichment of vitamin D target genes in GO kidney development terms, there is significant enrichment of vitamin A target genes in most of the gene sets we tested. Indeed, we observed that vitamin A target genes are enriched in CAKUT causal genes set, CAKUT differentially expressed genes set, kidney development pathways, the signaling by NOTCH pathway, and the GDNF/RET signaling axis pathway. There is also an overlap with the signaling by the WNT pathway, although it is not statistically significant. Overall, the significant overlaps observed between vitamin A and CAKUT gene sets indicate that vitamin A might be relevant to a broad range of urogenital abnormalities, pointing out the importance of nutritional management during pregnancy for fetal development. However, it should be clear that our study is dedicated to the generation of hypotheses. Additional studies are needed, using up-to-date datasets obtained with new omics techniques, to investigate the fine-grained effects of vitamin excess and deficiency on CAKUT, and to decipher the pathophysiological mechanisms. The increased availability of multi-omics technologies, such as whole genome sequencing and metabolomics will certainly open new directions, e.g. by revealing new causal genes or identifying metabolic disturbances due to environmental factors.
Zenodo: Overlap of vitamin A and vitamin D target genes with CAKUT-related processes. https://www.doi.org/10.5281/zenodo.4501623.32
This project contains the following underlying data in the ‘Data’ folder:
• VitA-CTD-Genes.txt (list of vitamin A target genes from CTD used for overlap analyses).
• VitA-Balmer2002-Genes.txt (list of vitamin A target genes from Balmer and Blomhoff used for overlap analyses).
• VitD-CTD-Genes.txt (list of vitamin D target genes from CTD used for overlap analyses).
• VitD-Ramagopalan2010.txt (list of vitamin D target genes from Ramagopalan et al. used for overlap analyses).
• PathwaysOfInterest.gmt (CAKUT-related gene sets used for overlap analyses).
• PathwaysOfInterestBackground.txt (list indicating which background GMT file to be used for the analysis of each CAKUT-related gene set).
• hsapiens.GO:BP.name.gmt (all gene sets from GO:BP used as background information for overlap analyses).
• hsapiens.REAC.name.gmt (all gene sets from Reactome used as background information for overlap analyses).
• hsapiens.WP.name.gmt (all gene sets from WikiPathways used as background information for overlap analyses).
This project contains the following underlying data in the ‘Result’ folder:
• VitA-CTD-Genes.csv (overlap analysis results for vitamin A targets from CTD).
• VitA-Balmer2002-Genes.csv (overlap analysis results for vitamin A targets from Balmer and Blomhoff).
• VitA-Merged.csv (merged table for the results presented in VitA-CTD-Genes.csv and VitA-Balmer2002-Genes.csv).
• VitD-CTD-Genes.csv (overlap analysis results for vitamin D targets from CTD).
• VitD-Ramagopalan2010.csv (overlap analysis results for vitamin D targets from Ramagopalan et al.).
• VitD-Merged.csv (merged table for the results presented in VitD-CTD-Genes.csv and VitD-Ramagopalan2010.csv).
Zenodo: Overlap of vitamin A and vitamin D target genes with CAKUT-related processes. https://www.doi.org/10.5281/zenodo.4501623.32
This project contains the following extended data:
This project contains the following extended data in the ‘Supp’ folder:
• CAKUT-CausalGenesPMID29079659-PMID30143558.txt (list of genes known to be mutated in the monogenic form of CAKUT).
• DEGsFromPMID30261159.txt (list of differentially expressed genes in CAKUT)
• SupplementaryAnalyses.odt (descriptions of the supplementary analyses).
• targetsFromDifferentSourcesOverlap.odt (contains the information on vitamin A and D targets from different sources (CTD, publications), presents source specific genes and common genes).
• VitA-Balmer2002-GeneMapping.csv (detailed table for the gene list from Balmer and Blomhoff).
This project contains the following extended data in the ‘Supp/CAKUTCausalGenesOverlapWithOthers’ folder:
• CAKUTCausalGenesOverlap.csv (overlap of CAKUT causal genes with other CAKUT-related gene sets without considering whether genes are vitamin targets or not).
This project contains the following extended data in the ‘Supp/RandomizedSampling’ folder:
• VitA-CTD-Genes-RS.csv (overlap analysis results for vitamin A targets from CTD using the randomized sampling approach)
• VitA-Balmer2002-Genes-RS.csv (overlap analysis results for vitamin A targets from Balmer and Blomhoff using the randomized sampling approach - please see answer to the reviewers).
• VitD-CTD-Genes-RS.csv (overlap analysis results for vitamin D targets from CTD using the randomized sampling approach).
• VitD-Ramagopalan2010-RS.csv (overlap analysis results for vitamin D targets from Ramagopalan et al. using the randomized sampling approach)
Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).
Ozisik O: Conceptualization, Data Curation, Formal Analysis, Project Administration, Writing – Original Draft Preparation; Ehrhart F: Conceptualization, Writing – Review & Editing; Evelo C: Conceptualization, Supervision, Writing – Review & Editing; Mantovani A: Conceptualization, Supervision, Writing – Original Draft Preparation; Baudot A: Conceptualization, Data Curation, Project Administration, Supervision, Writing – Original Draft Preparation.
We thank the members of European Joint Programme on Rare Diseases, in particular the members of the Environment/AOP Work Focus.
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Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Developmental toxicology
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Statistics, Systems Biology, Biological networks analysis and reconstruction
Is the work clearly and accurately presented and does it cite the current literature?
Yes
Is the study design appropriate and is the work technically sound?
Partly
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Partly
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
References
1. Jovanovic I, Zivkovic M, Kostic M, Krstic Z, et al.: Transcriptome-driven integrative exploration of functional state of ureter tissue affected by CAKUT.Life Sci. 2018; 212: 1-8 PubMed Abstract | Publisher Full TextCompeting Interests: No competing interests were disclosed.
Reviewer Expertise: Renal systems and computational biology.
Is the work clearly and accurately presented and does it cite the current literature?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Statistics, Systems Biology, Biological networks analysis and reconstruction
Is the work clearly and accurately presented and does it cite the current literature?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Human genetics, developmental biology, Wnt
Is the work clearly and accurately presented and does it cite the current literature?
Partly
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Developmental toxicology
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