Transcriptome-wide based identification of miRs in congenital anomalies of the kidney and urinary tract (CAKUT) in children: the significant upregulation of tissue miR-144 expression

The genetic cause of most CAKUT cases remains unknown. Urogenital tract and renal system phenotype depends on genetic, epigenetic and environmental factor interactions [27, 28]. It was shown that miRs could modulate genomic stability and mismatch repair [29]. More than 30 % of the human genes could be regulated by miRs [30] and miRs could represent biomarkers for environmental exposure [31]. In this study we applied the comprehensive bioinformatical approach in identification of miRs associated with human CAKUT. Predicted miRs were experimentally validated to be expressed in ureter tissue: hsa-miR-144, hsa-miR-375, hsa-miR-183 and hsa-miR-200a. We revealed a 5.7-fold induction of hsa-miR-144 in CAKUT samples compared to controls. Although it was suggested that elevated levels of hsa-miR-144 in body fluids could represent a biomarker of diabetic kidney injuries [32], the role and biomarker potential of this miR in CAKUT was not investigated.

The hsa-miR-144 gene is located in a polymorphic region (17q11.2) rich with CNVs, according to the database of genomic variants [33]. CNVs are functional variants which can alter the miR expression through the shift in miR gene dosage or the positioning of the miR gene regulatory elements [34]. Recent studies have investigated the association of CNVs with CAKUT and revealed that a significant proportion of patients harbor submicroscopic chromosomal imbalances, among others, in the 17q11-12 region [35, 36]. Therefore, hsa-miR-144 might be upregulated by specific CNV genotype. Long-range interactions between DNA segments affected by CNVs might also directly modify the miR expression pattern [34]. However, the mechanism responsible for miR expression regulation should not be interpreted solely through genetic variation due to the complexity of other epigenetical and environmental interactions [5, 37]. The inverse correlation pattern was described between hsa-miR-144 expression and genome-wide methylation status in cancer [38]. Epigenetic modifications have been postulated as a mechanism that facilitates the interaction between environmental factors and the genome during development, and its impact on disease susceptibility. As epigenetic modifications are reversible and susceptible to environmental stress (such as in utero environment), thus enabling developmental and temporal variability in gene expression patterns [5], these modifications could also lead to dysregulation of miR expression in early development, that could even remain postnatally. In mammals, multigenerational environmental effects have been documented by either epidemiological studies in human or animal experiments in rodents [39]. Steady state cellular miR levels represent the balance between miR biogenesis, influenced with described CNV and epigenetic mechanisms, and turnover [40]. However, the different turnover kinetics between certain miR isoforms and between different miRs is still not fully explained [40]. Therefore, future studies should also take into consideration, besides biogenesis, the turnover mechanisms and research of yet unknown molecular chaperones responsible for this process.

The development of the kidney and urinary tract is a complex process involving precise temporal and spatial modulation of gene expression responsible for correct proliferation, differentiation, and morphogenesis [41]. Analysis of the coexpression clusters of CoMeTa predicted hsa-miR-144 targets from the Co-Operational Level (COOL) analysis, and GO analysis of these clusters [14], revealed that a cluster of 511 putative targets was enriched with describes biological processes relevant for CAKUT pathology (Fig. 3). Certain GO biological processes enriched in the cluster (kidney development, urogenital system development, tube development, embryonic organ development) suggest that deregulated expression of hsa-miR-144 might contribute to impaired development of kidney and urinary tract. Although in this study we could not investigate the expression of hsa-miR-144 in human tissue during development, but after birth, certain developmental factors which represent crucial controllers of developmental stages could have prolonged activity in later stages after birth in CAKUT etiology. For example, TGF-?1, one of the main factors responsible for the kidney development, shows upregulation in human obstructive renal dysplasia [42] and prolonged expression in stenotic tissue of patients with UPJ obstruction [43]. In contrast with dysplastic tissues, normal prenatal and postnatal kidneys showed no significant TGF-?1 expression [42]. These data support the possibility of prolonged activity of certain miRs as important epigenetic factors in different stages of urogenital system development, where dysregulation of these miRs could lead to destabilization of important signaling pathways and to result in congenital malformations.

One of the main limitations of this study is the age difference between CAKUT patients and controls. However, miR-144 was found to be the sole miR that was consistently upregulated in the aging human and nonhuman primate cerebellum and in nonhuman primate cortex [44]. It was upregulated in the cerebromicrovascular endothelial cells of aged rats compared to young rats [45] and in skeletal muscle of old rhesus monkeys compared to young rhesus monkeys [46]. Although this highly conserved miR shows age dependent upregulation, this should not bias the results of our study in which hsa-miR-144 was upregulated in ureter samples of children with CAKUT compared to adult controls. Nevertheless, it is of importance to validate our results in age matched ureter tissue samples. Another limitation is the omitted kidney tissue. However, we deliberately selected the uniform tissue for expression analysis to improve the power of the study. Since the most common kind of surgical treatment in children with CAKUT is corrective surgery of the urinary tract, the human ureter tissue was selected as a target tissue for examination in this study. Ureter tissue has the same developmental origin as collecting duct system of the kidney, major and minor calyces and the renal pelvis [47], and the epithelium of the adult kidney consists of a number of specialized cell lineages where some originate from branching epithelial cells of the ureteric bud [41]. A drawback of our approach is that it assumes that the change in gene expression signature is solely the result of miR activity. It overlooks the activity of other transcription regulation factors. Another drawback is that the association of the miRs with CAKUT is based on miR target prediction. These could be the reasons for hsa-miR-183, hsa-miR-200a and hsa-miR-375 expression deviations from results of the CIA. However, the data about experimentally confirmed miR-mRNA interactions are still very scarce, thus the predictions still represents the most comprehensive approach. Although we used the advanced novel approach for filtering the prediction data, these results still need comprehensive experimental validation. Also, the discrepancy in the microarray platforms in our study and the one used for CoMeTa [14] might lead to overseeing the certain genes by the CIA based on CoMeTa miR target predictions, and thus influence the detection of miRs associated with CAKUT.