Systems biology approach to identify transcriptome reprogramming and candidate microRNA targets during the progression of polycystic kidney disease

Background: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation throughout the kidney parenchyma. It is caused by mutations in either of two genes, $PKD1$ and $PKD2$. Mice that lack functional $Pkd1 (Pkd1^{-/-})$, develop rapidly progressive cystic disease during embryogenesis, and serve as a model to study human ADPKD. Genome wide transcriptome reprogramming and the possible roles of micro-RNAs (miRNAs) that affect the initiation and progression of cyst formation in the $Pkd1^{-/-}$ have yet to be studied. miRNAs are small, regulatory non-coding RNAs, implicated in a wide spectrum of biological processes. Their expression levels are altered in several diseases including kidney cancer, diabetic nephropathy and PKD. Results: We examined the molecular pathways that modulate renal cyst formation and growth in the $Pkd1^{-/-}$ model by performing global gene-expression profiling in embryonic kidneys at days 14.5 and 17.5. Gene Ontology and gene set enrichment analysis were used to identify overrepresented signaling pathways in $Pkd1^{-/-}$ kidneys. We found dysregulation of developmental, metabolic, and signaling pathways (e.g. Wnt, calcium, $TGF-\beta$ and MAPK) in $Pkd^{-/-}$ kidneys. Using a comparative transcriptomics approach, we determined similarities and differences with human ADPKD: ~50% overlap at the pathway level among the mis-regulated pathways was observed. By using computational approaches (TargetScan, miRanda, microT and miRDB), we then predicted miRNAs that were suggested to target the differentially expressed mRNAs. Differential expressions of 9 candidate miRNAs, miRs-10a, -30a-5p, -96, -126-5p, -182, -200a, -204, -429 and -488, and 16 genes were confirmed by qPCR. In addition, 14 candidate miRNA:mRNA reciprocal interactions were predicted. Several of the highly regulated genes and pathways were predicted as targets of miRNAs. Conclusions: We have described global transcriptional reprogramming during the progression of PKD in the $Pkd1^{-/-}$ model. We propose a model for the cascade of signaling events involved in cyst formation and growth. Our results suggest that several miRNAs may be involved in regulating signaling pathways in ADPKD. We further describe novel putative miRNA:mRNA signatures in ADPKD, which will provide additional insights into the pathogenesis of this common genetic disease in humans

Metabolic Reprogramming and Reconstruction: Integration of Experimental and Computational Studies to Set the Path Forward in ADPKD

Metabolic reprogramming is a key feature of Autosomal Dominant Polycystic Kidney Disease (ADPKD) characterized by changes in cellular pathways occurring in response to the pathological cell conditions. In ADPKD, a broad range of dysregulated pathways have been found. The studies supporting alterations in cell metabolism have shown that the metabolic preference for abnormal cystic growth is to utilize aerobic glycolysis, increasing glutamine uptake and reducing oxidative phosphorylation, consequently resulting in ADPKD cells shifting their energy to alternative energetic pathways. The mechanism behind the role of the polycystin proteins and how it leads to disease remains unclear, despite the identification of numerous signaling pathways. The integration of computational data analysis that accompanies experimental findings was pivotal in the identification of metabolic reprogramming in ADPKD. Here, we summarize the important results and argue that their exploitation may give further insights into the regulative mechanisms driving metabolic reprogramming in ADPKD. The aim of this review is to provide a comprehensive overview on metabolic focused studies and potential targets for treatment, and to propose that computational approaches could be instrumental in advancing this field of research

Current epigenetic aspects the clinical kidney researcher should embrace

Chronic kidney disease (CKD), affecting 10-12% of the world's adult population, is associated with a considerably elevated risk of serious comorbidities, in particular, premature vascular disease and death. Although a wide spectrum of causative factors has been identified and/or suggested, there is still a large gap of knowledge regarding the underlying mechanisms and the complexity of the CKD phenotype. Epigenetic factors, which calibrate the genetic code, are emerging as important players in the CKD-associated pathophysiology. In this article, we review some of the current knowledge on epigenetic modifications and aspects on their role in the perturbed uraemic milieu, as well as the prospect of applying epigenotype-based diagnostics and preventive and therapeutic tools of clinical relevance to CKD patients. The practical realization of such a paradigm will require that researchers apply a holistic approach, including the full spectrum of the epigenetic landscape as well as the variability between and within tissues in the uraemic milieu

Emerging targeted strategies for the treatment of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a widespread genetic disease that leads to renal failure in the majority of patients. The very first pharmacological treatment, tolvaptan, received Food and Drug Administration approval in 2018 after previous approval in Europe and other countries. However, tolvaptan is moderately effective and may negatively impact a patient's quality of life due to potentially significant side effects. Additional and improved therapies are still urgently needed, and several clinical trials are underway, which are discussed in the companion paper Müller and Benzing (Management of autosomal-dominant polycystic kidney disease-state-of-the-art) Clin Kidney J 2018; 11: i2-i13. Here, we discuss new therapeutic avenues that are currently being investigated at the preclinical stage. We focus on mammalian target of rapamycin and dual kinase inhibitors, compounds that target inflammation and histone deacetylases, RNA-targeted therapeutic strategies, glucosylceramide synthase inhibitors, compounds that affect the metabolism of renal cysts and dietary restriction. We discuss tissue targeting to renal cysts of small molecules via the folate receptor, and of monoclonal antibodies via the polymeric immunoglobulin receptor. A general problem with potential pharmacological approaches is that the many molecular targets that have been implicated in ADPKD are all widely expressed and carry out important functions in many organs and tissues. Because ADPKD is a slowly progressing, chronic disease, it is likely that any therapy will have to continue over years and decades. Therefore, systemically distributed drugs are likely to lead to potentially prohibitive extra-renal side effects during extended treatment. Tissue targeting to renal cysts of such drugs is one potential way around this problem. The use of dietary, instead of pharmacological, interventions is another

Data-driven knowledge discovery in polycystic kidney disease

The use of data derived from genomics and transcriptomic to further develop our understanding of Polycystic Kidney Diseases and identify novel drugs for its treatment.LUMC / Geneeskund

Profiling of miRNAs and target genes related to cystogenesis in ADPKD mouse models

Autosomal polycystic kidney disease (ADPKD) is a common inherited renal disease characterized by the development of numerous fluid-filled cysts in both kidneys. We investigated miRNA-mediated regulatory systems and networks that play an important role during cystogenesis through integrative analysis of miRNA- and RNA-seq using two ADPKD mouse models (conditional Pkd1- or Pkd2-deficient mice), at three different time points (P1, P3, and P7). At each time point, we identified 13 differentially expressed miRNAs (DEmiRs) and their potential targets in agreement with cyst progression in both mouse models. These targets were involved in well-known signaling pathways linked to cystogenesis. More specifically, we found that the actin cytoskeleton pathway was highly enriched and connected with other well-known pathways of ADPKD. We verified that miR-182-5p regulates actin cytoskeleton rearrangement and promotes ADPKD cystogenesis by repressing its target genes-Wasf2, Dock1, and Itga4-in vitro and in vivo. Our data suggest that actin cytoskeleton may play an important role in renal cystogenesis, and miR-182-5p is a novel regulator of actin cytoskeleton and cyst progression. Furthermore, this study provides a systemic network of both key miRNAs and their targets associated with cyst growth in ADPKD.ope

Global microRNA profiling in human urinary exosomes reveals novel disease biomarkers and cellular pathways for Autosomal Dominant Polycystic Kidney Disease

MicroRNAs (miRNAs) play an important role in regulating gene expression in health and disease but their role in modifying disease expression in Autosomal Dominant Polycystic Kidney Disease (ADPKD) remains uncertain. Here, we profiled human urinary exosome miRNA by global small RNA-sequencing in an initial discovery cohort of seven patients with ADPKD with early disease (eGFR over 60ml/min/1.73m2), nine with late disease (eGFR under 60ml/min/1.73m2), and compared their differential expression with six age and sex matched healthy controls. Two kidney-enriched candidate miRNA families were identified (miR-192/miR-194-2 and miR-30) and selected for confirmatory testing in a 60 patient validation cohort by quantitative polymerase chain reaction. We confirmed that miR-192-5p, miR-194- 5p, miR-30a-5p, miR-30d-5p and miR-30e-5p were significantly downregulated in patient urine exosomes, in murine Pkd1 cystic kidneys and in human PKD1 cystic kidney tissue. All five miRNAs showed significant correlations with baseline eGFR and ultrasound-determined mean kidney length and improved the diagnostic performance (area under the curve) of mean kidney length for the rate of disease progression. Finally, inverse correlations of these two miRNA families with increased expression in their predicted target genes in patient PKD1 cystic tissue identified dysregulated pathways and transcriptional networks including novel interactions between miR-194-5p and two potentially relevant candidate genes, PIK3R1 and ANO1. Thus, our results identify a subset of urinary exosomal miRNAs that could serve as novel biomarkers of disease progression and suggest new therapeutic targets in ADPKD

Modulating Hallmarks of Cholangiocarcinoma

How are cholangiocarcinoma cells different from non-malignant cholangiocytes? All of the hallmarks of cancer apply- those reported in this dissertation include cell proliferation, migration, apoptosis, and evasion of growth suppression. My studies began with testing how apoptosis might be regulated through embelin, a small molecule reported to sensitize cells to apoptosis by blocking XIAP. My data however revealed that embelin reduced the proliferative capacity in cholangiocarcinoma cells, but did not increase cell death. Malignant cells exhibit dysregulation of microRNA processing and expression. Hence, my studies seeking ways that cholangiocarcinoma eludes apoptosis transitioned to the oncomiR miR-106b, which is overexpressed in cholangiocarcinoma. I observed that miR-106b protected cholangiocarcinoma cells from apoptosis. Genome-wide screening identified the landscape of miR-106b-targeted genes in a cholangiocarcinoma cell line, some with roles in tumor biology. MiR-106b targets included members of the Krüppel-like factor (KLF) family of transcription factors. The function of KLF2 in biliary epithelia or cholangiocarcinoma is unknown. I describe in part how the cholangiocyte senses the environment through the primary cilium and translates this to regulation of KLF2, a flow-responsive regulatory protein and tumor suppressor in several cancers. I observed lower expression of KLF2 in malignant cholangiocarcinoma cells and tumors compared to normal, and its enforced expression inhibited proliferation and migration while reducing sensitivity to apoptosis. In the normal bile duct epithelium, environmental cues are detected by the cholangiocyte primary cilium, a sensory organelle that functions as a signaling nexus for the cell. Cholangiocarcinoma cells are highly proliferative despite extracellular signals to remain quiescent. Cholangiocarcinoma cells often lose their cilia, resulting in altered communication and unchecked cell growth. I identified a cholangiocyte signaling axis in which the primary cilium maintains quiescence through enhanced KLF2 expression. I present the first finding of a ciliary-dependent KLF2 flow response in cholangiocytes. Overall, this dissertation sought deeper understanding of biochemical and molecular features of cholangiocarcinoma and added to our understanding of microRNAs and mechanosensory pathways

Hormonal regulation of microRNA expression in the ovary

The ovary is a dynamic organ that is charged with the responsibility of producing a viable gamete so that the circle of life can be reproduced for future generations. The ovary is also responsible for producing, secreting, and maintaining the proper hormone milieu of estrogens and progesterone for maintenance of pregnancy and the overall fitness of a woman's health. Understanding the mechanisms that regulate the interplay between hormone action and biological function is critical for furthering our knowledge of fertility and reproductive health. For decades, research has been conducted on understanding the transcriptional regulation of ovarian gene expression and how this relates to reproductive function. Recently, attention has turned to alternative forms of gene regulation, including post-transcriptional gene regulation. One mechanism of post-transcriptional gene regulation is the expression and function of microRNA (miRNA). These highly conserved, short, non-coding RNA molecules primarily silence gene expression by directly interfering with protein translation or causing the degradation of messenger RNA. The focus of these studies was to first determine if miRNA are necessary for female fertility. Conditional deletion of Dicer, a key processing enzyme in miRNA biogenesis, in ovarian granulosa cells, the oviduct, and uterus, led to a drastic decrease in ovulation rate and complete infertility in female mice. To further investigate the role of miRNA in ovulation, we next investigated miRNA-212 and -132. While these two co-transcribed miRNA were highly induced by the luteinizing hormone surge immediately prior to ovulation, they did not appear to have an effect on female fertility in the mouse. In a second series of studies, we analyzed the regulation of miRNA by hormones in two in vivo models. We found that miRNA expression was altered in theca cells from women suffering from polycystic ovarian syndrome (PCOS) and that expression of miRNA was altered in the fetal ovaries of sheep exposed to an excess of prenatal androgens. Taken together, these studies provide evidence that miRNA are crucial for female fertility and ovarian function and that hormones influence the expression of ovarian miRNA in diseased states. These studies support the need for further study to understand the mechanisms through which these post-transcriptional regulators affect ovarian function, so that we can potentially use them as a therapeutic target to help overcome infertility and/or disease

miRNAs in kidney disease

A l'heure actuelle, les microARNs sont reconnus comme des régulateurs essentiels de l'expression des protéines. Des anomalies dans leur fonction sont associées au développement de nombreuses pathologies. Ainsi, de nombreuses études s'intéressent au potentiel des microARNs en tant que biomarqueurs ou cibles thérapeutiques dans une grande variété de pathologies. De plus, un certain nombre de recherches font le lien entre microARNs et pathologies rénales et soulignent l'intérêt des microARNs en tant que biomarqueurs pour améliorer le diagnostic et le pronostic des maladies rénales en utilisant des échantillons sanguins, sériques, de tissus et d'urine. Dans le cadre de cette thèse, nous avons étudié :1) L'association des microARNs urinaires avec l'évolution de la maladie rénale chronique (MRC) chez l'adulte. La prévalence de la MRC est actuellement estimée à 5-10% de la population et est en constante augmentation. La détection précoce et l'identification de patients ayant une MRC progressant rapidement vers l'insuffisance rénale sont la clé pour une meilleure prise en charge de ces patients. Actuellement les outils non-invasifs comme l'albuminurie ou l'estimation du débit de filtration glomérulaire manquent de précision. Dans notre travail, nous avons tenté d'identifier les modifications urinaires des microRNAs afin d'identifier de nouveaux biomarqueurs non-invasifs associés à la progression de la MRC. Nous avons analysé les modifications des microARNs urinaires par séquençage à haut débit dans des échantillons d'urine de 70 patients atteints de MRC et corrélé leurs profils d'expression à la progression de la maladie. Cela a amené à l'identification de 25 microARNs urinaires (pvalue ajustée <0.05) potentiellement associés à la progression de la MRC. Parmi ceux-là, quatre microARNs (hsa-miR-34c-5p, hsa-miR-410-3p, hsa-miR-301b-3p, and hsa-miR-145-5p) ont été sélectionnés pour être validés dans une cohorte indépendante de 52 patients atteints de MRC. L'augmentation de l'abondance urinaire de hsa-miR-145-5p a été confirmée comme étant associée à la progression de la MRC. Des analyses in vitro de l'effet de l'inhibition de hsa-miR-145-5p dans les cellules rénales ont mis en évidence que ce microARN semblait être impliqué dans le processus de nécrose. En conclusion, cette étude nous a permis d'identifier hsa-miR-145-5p comme marqueur potentiel de la progression de la MRC. 2) La présence de microARNs urinaires associés à la néphropathie obstructive, une maladie fréquemment rencontrée chez les enfants qui peut conduire, dans les cas graves, à l'insuffisance rénale précoce. Dans cette étude, nous avons utilisé la biologie des systèmes et avons combiné des données microARN et ARNm de néphropathie obstructive humaine et animale pour obtenir des informations sur les mécanismes possibles impliqués dans cette maladie. En particulier, nous avons étudié simultanément le miRNome urinaire de nourrissons présentant une obstruction de la jonction pyélo-urétérale et le miRNome et le transcriptome tissulaire rénal chez la souris dans le modèle animal d'obstruction urétérale unilatéral (OUU) partiel et néonatal. Plusieurs centaines de microARNs et d'ARNms étant modifiés, la combinaison des microARNs des deux espèces avec les ARNms cibles associés a permis de sélectionner les 5 microARNs et 35 ARNms les plus fortement associés à la néphropathie obstructive. Une validation in vitro et in vivo a mis en avant que let-7a-5p et miR-29-3p ainsi que deux nouvelles cibles potentielles, l'E3 ubiquitin-protein ligase (DTX4) et neuron navigator 1 (NAV1) étaient dérégulées au cours de cette pathologie. Cette étude est la première à corréler le modèle animal d'OUU partiel et néonatal avec l'obstruction pyélo-urétérale chez l'Homme dans une analyse intégrée de biologie des systèmes. Nos résultats ont révélé let-7a et miR-29b en tant que molécules potentiellement impliquées dans le développement de la fibrose dans la néphropathie obstructive via le contrôle de DTX4 chez l'homme et la souris, ce qui n'aurait pas été identifiable autrement.MicroRNAs are now recognized as key players in the regulation of proteins and any abnormality in their function is a cause for pathway instability, leading to pathological conditions. Numerous reports from a variety of pathologies provide new data about microRNAs function, their targets and their potential as biomarkers and possible ways to control microRNAs' expression for potential therapeutic purpose. A number of reports also connect microRNAs with pathological conditions in the kidney and point to the use of microRNAs as biomarkers for diagnosis and prognosis of kidney disease in blood, serum, tissue and urine samples. In this thesis, we researched:1) A possible role of the microRNAs in the progression of adult chronic kidney disease (CKD), a disease representing a global burden with the tendency to rise worldwide. Progression of CKD is still very hard to detect non-invasively with the currently used clinical tools (eGFR and albuminuria). In our work we studied alterations of the level of the microRNAs in human urine samples of patients with fast or slow progression of CKD, in order to identify new potential biomarkers for non-invasive progression of CKD. Using Next Generation Sequencing, we analyzed urinary microRNA modifications in urine samples of 70 patients with established CKD and correlated their expression profiles to disease progression. This lead to the identification of 25 urinary microRNAs significantly associated to CKD progression (adjusted pvalue<0.05). Among those, four microRNAs (hsa-miR-34c-5p, hsa-miR-410-3p, hsa-miR-301b-3p, and hsa-miR-145-5p) were selected for validation in an independent cohort of 52 patients with CKD. Increased urinary abundance of hsa-miR-145-5p was confirmed to be associated to progression of CKD. In vitro exploration of the effects of hsa-miR-145-5p inhibition in human kidney cells showed that the microRNA seemed to be involved in necrotic processes. In conclusion we have identified hsa-miR-145-5p as potential urinary microRNA marker of CKD progression. 2) The identification of microRNAs associated to obstructive nephropathy, a frequently encountered disease in children that can lead, in severe cases, to end stage renal disease (ESRD). In this study we used a comprehensive system biology analysis in which we combined micro- and mRNA data from human and animal obstructive nephropathy to obtain information on possible mechanisms involved in this disease. In particular, we have studied in parallel the urinary miRNome of infants with ureteropelvic junction (UPJ) obstruction and the kidney tissue miRNome and transcriptome of the corresponding neonatal partial unilateral ureteral obstruction (UUO) mouse model. Several hundreds of microRNAs and mRNAs displayed changed abundance during disease. Combination of microRNAs in both species and associated mRNAs let to the prioritization of 5 microRNAs and 35 mRNAs associated to disease. In vitro and in vivo validation identified consistent dysregulation of let-7a-5p and miR-29-3p and new potential targets, E3 ubiquitin-protein ligase (DTX4) and neuron navigator 1 (NAV1). Our study is the first to correlate a mouse model of neonatal partial UUO with human UPJ obstruction in a comprehensive systems biology analysis. Our data revealed let-7a and miR-29b as molecules potentially involved in the development of fibrosis in UPJ obstruction via the control of DTX4 in both man and mice that would not be identified otherwise