BACKGROUND
congenital obstructive nephropathy is the main cause of end stage renal disease  in children  <cit> .
this contrasts sharply with adult esrd, which for the greater part originates from type ii diabetes and hypertension  <cit> .
the most frequent cause of congenital urinary tract obstruction is ureteropelvic junction  obstruction with an estimated incidence of one in 1000– <dig>  <cit> .
the spectrum of renal abnormalities varies greatly in upj obstruction ranging from subtle changes such as modified proximal or tubular size, chronic tubulointerstitial injury, glomerulosclerosis, fibrosis, aberration of nephron development and in severe cases  renal dysplasia  <cit> .
the gold standard in diagnosis of upj is by prenatal ultrasonography with subsequent evaluation in the postnatal period  <cit> .
however, this method is not sensitive enough to accurately estimate renal function and functioning nephron number  <cit> .
this has led to an urgent need for the development of biomarkers to assess the severity of upj obstruction and to help the clinicians to decide if and when pyeloplasty is required  <cit> .
due to the fact that limited human kidney samples are available, almost all observations on the pathophysiology of upj obstruction are based on animal models, which potentially limit the transferability of the observations in the clinical context.
as a consequence, the pathophysiological mechanisms of upj obstruction remain incompletely understood.
renal lesions in upj obstruction have been described including tubular proliferation/apoptosis, renin-angiotensin system activation, inflammation, and fibrosis  <cit> .
interstitial fibrosis is a late consequence of congenital upj obstruction, and can be attenuated by early release of obstruction, but not if nephron number is reduced  <cit> .
it is becoming increasingly clear that patients with congenital urinary tract obstruction must be followed into adulthood, as the lesions can progress over the entire life  <cit> .
micrornas  are small non-coding rnas  that regulate gene expression by blocking the translation of proteins and are involved in multiple molecular pathways and pathologies.
mirnas are now considered promising molecules for biomarkers and/or targeted therapy of disease .
while, kidney diseases are no exception  <cit> , to our knowledge no studies specifically report mirnas related to upj obstruction.
there is evidence connecting dysregulated mirnas including mir- <dig> and mir- <dig> with kidney fibrosis , an important feature in severe upj obstruction.
moreover, knock-down of dicer  leads to congenital anomalies of the kidney and the urinary tract  in mice  <cit> .
mirnas are very stable in urine, a biofluid which can be collected non-invasively and can be valuable source of molecules to monitor diseases of the kidney and urinary tract  <cit> .
these observations suggest that studying mirnas in upj obstruction might help to understand the development of obstructive nephropathy and/or provide early markers of pathological obstruction.
in this study, we analysed the modification of urinary mirnas in upj obstruction.
to improve upon the clinical translatability of our results and compensate for the lack of tissue availability in human disease, we combined mirna data obtained in human urine and mirna and mrna data in kidney tissue of a neonatal partial unilateral ureteral obstruction  mouse model.
this combined systems biology-based approach followed by an in vitro validation pointed to the consistent dysregulation of specific mirnas, let-7a-5p and mir-29-3p and to new potential targets, e <dig> ubiquitin-protein ligase  and neuron navigator  <dig>  in upj obstruction that would not be identified otherwise.
RESULTS
mirna abundance changes in urine of newborns with upj obstruction
a total of  <dig> male upj obstruction patients and eight male healthy age-matched  individuals were studied .
the severity of the obstruction varied with hydronephrosis grades from  <dig> to  <dig> and pelvic dilatation sizes from  <dig> to 40 mm .
we compared the urinary mirna abundance of upj obstruction patients to urine of healthy controls using microarray analysis.
this yielded the identification of  <dig> mirnas with changed urinary abundance between the two groups .table  <dig> clinical data of the human upj obstruction patients
n
hn hydronephrosis, n.a.
not applicable
mirna and mrna expression changes in renal tissue of a neonatal partial uuo mouse model
the renal mirna and mrna profiles of nine neonatal mice with partial uuo  and nine control sham operated mice were studied using microarray analysis .
this led to the identification of  <dig> differentially expressed mirnas and  <dig> differentially expressed mrnas, respectively .table  <dig> experimental data of the partial uuo model animals
hn hydronephrosis, n.a.
not applicable
identification of most prominent dysregulated mirnas commonly associated with obstructive nephropathy in humans and mice
to prioritize the molecular features with potentially the highest impact on the development of kidney lesions, we next identified the mirnas that could consistently reflect the human disease by comparing the similarity of the human  and animal  mirna signature taking advantage from the fact that mirnas are well conserved between species  <cit> .
the  <dig> differentially expressed human mirnas and  <dig> differentially expressed mouse mirnas were combined.
this led to the identification of  <dig> common mirnas  that were reduced to five mirnas when applying a fold change threshold of  <dig>  in mice tissue.
these five mirnas were let-7a-5p mir-16-5p, mir-29b-3p, mir-125b-5p and mir-26a-5p .
correlation of the urinary abundance of the five mirnas in upj patients with clinical parameters showed that mir-125-5p was inversely correlated with hydronephrosis grade .
in addition, a slight but significant inverse correlation with pelvic diameter was also observed for mir-let-7a-5p and mir-125-5p and with hydronephrosis grade for mir-let-7a-5p and mir-26a-5p, and a slight but significant positive correlation with age for mir-let-7a-5p and mir-16-5p .table  <dig> most prominent dysregulated mirnas commonly associated to the partial uuo model and human upj obstruction
fc fold change
identification of most prominent dysregulated pathways and mrna targets in obstructive nephropathy
seven hundred six differentially expressed mrnas were observed in kidneys of neonatal mice with partial uuo, including increased expression of markers of fibrosis such as tgf-β <dig> and different forms of collagen .
to prioritize the mrna targets, the five selected common mirnas in mice and human obstructive nephropathy were combined with these  <dig> differentially expressed mrnas using ingenuity pathway analysis .
then, we focused on the direct connections of the five mirnas as generated from the predicted networks from ipa analysis.
this led to the identification of  <dig> predicted target mrnas for these five mirnas .
next, in order to focus on the most prominent molecular changes, only mrnas predicted to be targeted by at least two of the five mirnas were kept for further analysis: e <dig> ubiquitin-protein ligase dxt <dig> , leiomodin- <dig> , a disintegrin-like and metallopeptidase  with thrombospondin type  <dig> motif,  <dig>  and neuron navigator  <dig>  .table  <dig> most prominent deregulated mrna targets in obstructive nephropathy
gene symbol
dtx4
lmod1
adamts19
nav1
fc fold change
mirna knockdown leads to dysregulated expression of the mrna targets in renal cells in vitro
to validate the results of the in silico analysis, we next assessed whether the predicted mirna-mrna target pairs were directly associated in human renal cells.
human kidney cells  were treated for 48 h with chemically modified molecules blocking the action of specific mirna .
in the presence of antagomirs, the detected signal of let-7a, mir-16-5p, mir-125b-5p, mir-26a-5p and mir-29b-3p was significantly decreased .fig.
<dig> selected mirna expression in response to antagomirs in vitro.
expression of let-7a , mir-125b-5p , mir-16-5p , mir-26a-5p  and mir-29b-3p  was assessed by rt-pcr in hk <dig> cells treated or not with antagomirs.
cont: control; antag: antagomir.
*p <  <dig>  versus cont
mrna expression of let-7a and mir-125b-3p target dtx <dig> was significantly increased in response to downregulation of let-7a but was unmodified by antagomir anti-mir-125b-3p .
moreover, significant upregulation of neuron navigator  <dig> nav <dig> was observed in hk <dig> cells treated with the mir-29b-3p antagomir .
surprisingly, the use of antagomirs for mir-125b-5p and mir-26a-5p resulted in slight but significant downregulation of lmod <dig>  adamts <dig> and nav <dig> , a result opposite than the predicted regulation, which possibly indicates an indirect mechanism of effect of these two mirnas on these targets.
antagomirs for mir-125b-5p, mir-16-5p and mir-29b-3p showed no effect on dtx <dig>  lmod <dig> and adamts <dig>  respectively .fig.
<dig> selected mrna predicted target expression in response to antagomirs in vitro.
expression of e <dig> ubiquitin-protein ligase dxt <dig>  , leiomodin- <dig>  , a disintegrin-like and metallopeptidase  with thrombospondin type  <dig> motif,  <dig>   and neuron navigator  <dig>   was assessed by rt-pcr in hk <dig> cells treated or not with antagomirs against let-7a, mir-125b-5p, mir-16-5p, mir-26a-5p or mir-29b-3p.
cont: control; antag: antagomir.
*p <  <dig>  versus cont
dtx <dig> and nav <dig> are dysregulated during complete uuo in vivo
since obstructive nephropathy potentially induces different pathways in the developing and the adult kidney we also verified the expression of the mrna targets in the adult mouse complete uuo model, using eight male mice with complete uuo and six sham operated mice.
we demonstrated that, similarly to what we observed in partial uuo in neonatal mice, dtx <dig> and nav <dig> expression was significantly increased in uuo mice compared to the sham , while lmod <dig> was not modified .
the signal for adamts <dig> was too weak to provide any reliable data.
in addition to the targets, we also showed that the renal expression of tgfβ and collagen  <dig>  two markers of fibrosis, and il- <dig>  a marker of inflammation was significantly increased in uuo mice compared to the sham , validating the pathological changes observed in this model.fig.
<dig> selected mrna predicted target expression in response to adult complete uuo in vivo.
renal expression of e <dig> ubiquitin-protein ligase dxt <dig>  , leiomodin- <dig>  , neuron navigator  <dig>  , transforming growth factor beta  , collagen  <dig>  , and interleukin  <dig>   was assessed by rt-pcr in adult mice after 7 days uuo.
*p <  <dig>  versus sham
discussion
obstructive nephropathies, with upj obstruction as the prototypic obstructive nephropathy, are frequently encountered developmental anomalies in the pediatric population.
although renal fibrosis and inflammation are involved in the pathophysiology of severe upj obstruction, the molecular mechanisms underlying evolution of the lesions remain undetermined.
in addition, most of this information has been obtained in animal models of disease.
recently discovered small non-coding rnas called mirnas are excreted and are stable in urine.
hence, urinary mirnas could help to further decipher the pathophysiology of upj obstruction and generate the missing link between human and animal data.
in this study we combined human urinary mirna data and animal kidney tissue mirna and mrna data in a systems biology approach to obtain insight in the pathophysiology of the disease and improve upon the translatability of the results.
the combined approach and independent validation pointed to the consistent dysregulation of specific mirnas and to new potential targets in upj obstruction.
mirnas let-7a-5p, mir-125b-5p, mir-16-5p, mir-26a-5p and mir-29b-3p were consistently modified in mice and humans.
among these, mir-29b is a well-known player in renal pathologies and especially fibrosis.
indeed, mir-29b targets specific fibrotic molecules including collagens or α-smooth muscle actin, and its abundance is reduced in many fibrotic pathologies as its expression is inhibited by tgfβ  <cit> .
the protective role of mir-29b in fibrosis was further demonstrated in vivo since restoring mir-29b levels in a diabetic nephropathy animal model reversed accumulation of renal extracellular matrix  <cit> .
on the other hand, to our knowledge, this is the first time that mir- <dig> and let-7a are associated to the development of kidney disease, as these mirnas were mostly characterized to be involved in cancer .
downregulation of let-7a has also been observed in scleroderma, contributing to the excessive deposition of collagen and tissue fibrosis in the skin  <cit> .
few data is available for mir-125b and kidney disease.
circulating mir-125b was found downregulated in chronic kidney disease  in hypertensive patients suffering from cardiovascular disease  <cit> .
in another study upregulation of mir-125b protected against cisplatin-induced kidney injury via inhibition of nuclear factor erythroid-2-related factor  <dig>  <cit> .
mir-26a has previously been reported to be over expressed in lung epithelia during development and to be involved in glomerular and tubular injury related to podocyte damage and maintenance of glomerular filtration rate  <cit> .
a recent small-scale study  identified in kidney tissue five mirnas associated to upj obstruction.
none of those overlapped with the human urinary mirnas in our study and one, mir-342-5p, was also found in kidney tissue to be associated to obstruction in the partial neonatal mouse model, however with an opposite regulation  <cit> .
a total of  <dig> putative mrna targets for these differentially expressed mirnas were predicted with the use of ingenuity pathway analysis.
in vitro validation of the predicted mirna-mrna pairs and in vivo assessment of the observed mrna changes led to the sound confirmation of the regulation of e <dig> ubiquitin-protein ligase dtx <dig> by let-7a.
although e <dig> ubiquitin-protein ligase dtx <dig> has not yet been found associated to obstructive nephropathy, using the kidney & urinary pathway knowledge base  we observed that it has been found to be induced in models of polycystic kidney disease  <cit>  and in a lupus nephritis model  <cit> .
furthermore, dtx <dig> is a member of notch signaling non-canonical pathway  <cit> .
it has been described that activation of notch signaling can lead to fibrosis .
hence one can hypothesize that downregulation of let-7a, activates dtx <dig> and the notch signaling pathway, promoting the progression of fibrosis in upj obstruction.
nav <dig> is a protein mostly found in neurons and is reported to play a critical role in microtubule development  <cit> , but has not yet been implicated in upj obstruction.
coinciding with dtx <dig>  nav <dig> expression is also induced in a model of polycystic kidney disease  <cit>  and is induced in vitro by the major pro-fibrotic cytokine tgfβ  <cit> , potentially linking nav <dig> induction to fibrosis.
it is notable, though, that the regulation of nav <dig> by mir-29b-3p did not follow the classical regulation pattern  in the partial uuo model.
in contrast, in the in vitro experiment nav <dig> followed the predicted regulation and confirming that it may be a direct target of mir-29b.
previous reports have documented that some mirnas may not target mrnas directly, but only block the protein translation, leaving the mrna intact .
furthermore, other reports point to the fact that mirnas may induce the same direction of regulation of their mrna targets depending on the timeframe and conditions , providing an explanation of a possible indirect mechanism of mir-29b-nav <dig> regulation.
nevertheless, further investigation is needed to determine if nav <dig> is a direct target of mir-29b in vivo and if it is an interesting molecule in the context of upj obstruction.
combination of the human urine mirna data with the mouse tissue data increased the confidence in the human data and allowed selecting the most promising mirnas in human disease.
a downside of our approach is that this type of prioritization will only focus on part of the molecular mechanisms of upj.
if signals for specific mechanisms are absent in, for example, humans due to technical limitations or absence of shedding of mirnas in urine but not in mouse tissue, this mechanism is not necessarily irrelevant in the pathophysiology of upj.
another shortcoming of our study is the fact that we used unadjusted p-values.
infants with upj obstruction included in the study displayed a wide range of hydronephrosis  and pelvic diameter  which generates considerable variability in the upj obstruction group.
the mean fold change after comparison of healthy controls and upj obstruction patients was only  <dig>  fold  in this data.
this is probably due to the fact that during excretion/shedding of mirnas in urine the in-situ mirna changes are flattened out.
moreover, correction for multiple testing resulted in no significant mirnas in humans.
we therefore continued the prioritization with unadjusted p values but we compensated for this shortcoming by the independent validation of the selected targets.
finally, the question remains whether observations in mouse tissue or human urine are faithfully reflecting the changes in the human kidney.
in the past, studies have used a similar approach as ours, i.e.
by combining urine and tissue analysis, and/or by combining human and animal data, to understand the role of mirnas in the development of renal lesions.
for example, mir- <dig> has been found to be consistently increased in animal and human samples of both urine and kidney tissue in the context of renal fibrosis .
however, during acute kidney injury, mir- <dig> was increased in kidney tissue and decreased in the urine in a rat model, and increased in human urine  <cit> .
in a study of wang et al.
again in a kidney injury model, mir-10a and mir-30d were found decreased in tissue and increased in urine after injury  <cit> .
finally, mir- <dig> was observed in different studies to be up-regulated in tissue in animal model of fibrosis, but down-regulated in human tissue samples and increased in human urine samples .
these examples show that there is no common regulation pattern for mirnas in urine and kidney and in different species, even in the same pathological context and despite the fact that their role in renal lesions has been established with confidence.
but by taking advantage of the conservation ability of mirnas, combination of urine and tissue analysis, and/or human and animal data allows to identify features that are consistently and significantly modified as being associated with the disease with more confidence and can help transfer observations from animal models to human research.
in our study, we did not observe enriched canonical pathways when studying the mirnas of human or mouse upj obstruction separately even if around hundreds of mirnas were found significantly different.
hence we have used both statistical selection, combination of different omics data  and pathway enrichment analysis to identify mirnas and their targets most likely involved in the etiology of upj obstruction.
CONCLUSIONS
collectively this 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 dtx <dig> in both man and mice that would not be identified otherwise.
we believe that our approach of combining omics data is generally applicable.
many omics studies generate long lists of differentially expressed molecules that are difficult to prioritize and does not necessarily inform on the actual impact of these changes in disease.
to further improve on the validity and clinical translatability of the data, and because urine is a rich source of biomarkers that can be collected easily and non-invasively, combination of human urine and animal tissue data  could be of great help to better understand the molecular mechanisms involved in the development of many complex diseases.

 congenital obstructive nephropathy is the main cause of end stage renal disease in children. this contrasts sharply with adult esrd, which for the greater part originates from type ii diabetes and hypertension. the most frequent cause of congenital urinary tract obstruction is ureteropelvic junction obstruction with an estimated incidence of one in 1000–. the spectrum of renal abnormalities varies greatly in upj obstruction ranging from subtle changes such as modified proximal or tubular size, chronic tubulointerstitial injury, glomerulosclerosis, fibrosis, aberration of nephron development and in severe cases renal dysplasia. the gold standard in diagnosis of upj is by prenatal ultrasonography with subsequent evaluation in the postnatal period. however, this method is not sensitive enough to accurately estimate renal function and functioning nephron number. this has led to an urgent need for the development of biomarkers to assess the severity of upj obstruction and to help the clinicians to decide if and when pyeloplasty is required. due to the fact that limited human kidney samples are available, almost all observations on the pathophysiology of upj obstruction are based on animal models, which potentially limit the transferability of the observations in the clinical context. as a consequence, the pathophysiological mechanisms of upj obstruction remain incompletely understood. renal lesions in upj obstruction have been described including tubular proliferation/apoptosis, renin-angiotensin system activation, inflammation, and fibrosis. interstitial fibrosis is a late consequence of congenital upj obstruction, and can be attenuated by early release of obstruction, but not if nephron number is reduced. it is becoming increasingly clear that patients with congenital urinary tract obstruction must be followed into adulthood, as the lesions can progress over the entire life. micrornas are small non-coding rnas that regulate gene expression by blocking the translation of proteins and are involved in multiple molecular pathways and pathologies. mirnas are now considered promising molecules for biomarkers and/or targeted therapy of disease. while, kidney diseases are no exception, to our knowledge no studies specifically report mirnas related to upj obstruction. there is evidence connecting dysregulated mirnas including mir- and mir- with kidney fibrosis, an important feature in severe upj obstruction. moreover, knock-down of dicer leads to congenital anomalies of the kidney and the urinary tract in mice. mirnas are very stable in urine, a biofluid which can be collected non-invasively and can be valuable source of molecules to monitor diseases of the kidney and urinary tract. these observations suggest that studying mirnas in upj obstruction might help to understand the development of obstructive nephropathy and/or provide early markers of pathological obstruction. in this study, we analysed the modification of urinary mirnas in upj obstruction. to improve upon the clinical translatability of our results and compensate for the lack of tissue availability in human disease, we combined mirna data obtained in human urine and mirna and mrna data in kidney tissue of a neonatal partial unilateral ureteral obstruction mouse model. this combined systems biology-based approach followed by an in vitro validation pointed to the consistent dysregulation of specific mirnas, let-7a-5p and mir-29-3p and to new potential targets, e ubiquitin-protein ligase and neuron navigator in upj obstruction that would not be identified otherwise.  mirna abundance changes in urine of newborns with upj obstruction a total of male upj obstruction patients and eight male healthy age-matched individuals were studied. the severity of the obstruction varied with hydronephrosis grades from to and pelvic dilatation sizes from to 40 mm. we compared the urinary mirna abundance of upj obstruction patients to urine of healthy controls using microarray analysis. this yielded the identification of mirnas with changed urinary abundance between the two groups.table clinical data of the human upj obstruction patients n hn hydronephrosis, n.a. not applicable mirna and mrna expression changes in renal tissue of a neonatal partial uuo mouse model the renal mirna and mrna profiles of nine neonatal mice with partial uuo and nine control sham operated mice were studied using microarray analysis. this led to the identification of differentially expressed mirnas and differentially expressed mrnas, respectively.table experimental data of the partial uuo model animals hn hydronephrosis, n.a. not applicable identification of most prominent dysregulated mirnas commonly associated with obstructive nephropathy in humans and mice to prioritize the molecular features with potentially the highest impact on the development of kidney lesions, we next identified the mirnas that could consistently reflect the human disease by comparing the similarity of the human and animal mirna signature taking advantage from the fact that mirnas are well conserved between species. the differentially expressed human mirnas and differentially expressed mouse mirnas were combined. this led to the identification of common mirnas that were reduced to five mirnas when applying a fold change threshold of in mice tissue. these five mirnas were let-7a-5p mir-16-5p, mir-29b-3p, mir-125b-5p and mir-26a-5p. correlation of the urinary abundance of the five mirnas in upj patients with clinical parameters showed that mir-125-5p was inversely correlated with hydronephrosis grade. in addition, a slight but significant inverse correlation with pelvic diameter was also observed for mir-let-7a-5p and mir-125-5p and with hydronephrosis grade for mir-let-7a-5p and mir-26a-5p, and a slight but significant positive correlation with age for mir-let-7a-5p and mir-16-5p.table most prominent dysregulated mirnas commonly associated to the partial uuo model and human upj obstruction fc fold change identification of most prominent dysregulated pathways and mrna targets in obstructive nephropathy seven hundred six differentially expressed mrnas were observed in kidneys of neonatal mice with partial uuo, including increased expression of markers of fibrosis such as tgf-β and different forms of collagen. to prioritize the mrna targets, the five selected common mirnas in mice and human obstructive nephropathy were combined with these differentially expressed mrnas using ingenuity pathway analysis. then, we focused on the direct connections of the five mirnas as generated from the predicted networks from ipa analysis. this led to the identification of predicted target mrnas for these five mirnas. next, in order to focus on the most prominent molecular changes, only mrnas predicted to be targeted by at least two of the five mirnas were kept for further analysis: e ubiquitin-protein ligase dxt, leiomodin-, a disintegrin-like and metallopeptidase with thrombospondin type motif, and neuron navigator.table most prominent deregulated mrna targets in obstructive nephropathy gene symbol dtx4 lmod1 adamts19 nav1 fc fold change mirna knockdown leads to dysregulated expression of the mrna targets in renal cells in vitro to validate the results of the in silico analysis, we next assessed whether the predicted mirna-mrna target pairs were directly associated in human renal cells. human kidney cells were treated for 48 h with chemically modified molecules blocking the action of specific mirna. in the presence of antagomirs, the detected signal of let-7a, mir-16-5p, mir-125b-5p, mir-26a-5p and mir-29b-3p was significantly decreased.fig. selected mirna expression in response to antagomirs in vitro. expression of let-7a, mir-125b-5p, mir-16-5p, mir-26a-5p and mir-29b-3p was assessed by rt-pcr in hk cells treated or not with antagomirs. cont: control; antag: antagomir. *p <  versus cont mrna expression of let-7a and mir-125b-3p target dtx was significantly increased in response to downregulation of let-7a but was unmodified by antagomir anti-mir-125b-3p. moreover, significant upregulation of neuron navigator nav was observed in hk cells treated with the mir-29b-3p antagomir. surprisingly, the use of antagomirs for mir-125b-5p and mir-26a-5p resulted in slight but significant downregulation of lmod adamts and nav, a result opposite than the predicted regulation, which possibly indicates an indirect mechanism of effect of these two mirnas on these targets. antagomirs for mir-125b-5p, mir-16-5p and mir-29b-3p showed no effect on dtx lmod and adamts respectively.fig. selected mrna predicted target expression in response to antagomirs in vitro. expression of e ubiquitin-protein ligase dxt, leiomodin-, a disintegrin-like and metallopeptidase with thrombospondin type motif, and neuron navigator was assessed by rt-pcr in hk cells treated or not with antagomirs against let-7a, mir-125b-5p, mir-16-5p, mir-26a-5p or mir-29b-3p. cont: control; antag: antagomir. *p <  versus cont dtx and nav are dysregulated during complete uuo in vivo since obstructive nephropathy potentially induces different pathways in the developing and the adult kidney we also verified the expression of the mrna targets in the adult mouse complete uuo model, using eight male mice with complete uuo and six sham operated mice. we demonstrated that, similarly to what we observed in partial uuo in neonatal mice, dtx and nav expression was significantly increased in uuo mice compared to the sham, while lmod was not modified. the signal for adamts was too weak to provide any reliable data. in addition to the targets, we also showed that the renal expression of tgfβ and collagen two markers of fibrosis, and il- a marker of inflammation was significantly increased in uuo mice compared to the sham, validating the pathological changes observed in this model.fig. selected mrna predicted target expression in response to adult complete uuo in vivo. renal expression of e ubiquitin-protein ligase dxt, leiomodin-, neuron navigator, transforming growth factor beta, collagen, and interleukin was assessed by rt-pcr in adult mice after 7 days uuo.*p <  versus sham discussion obstructive nephropathies, with upj obstruction as the prototypic obstructive nephropathy, are frequently encountered developmental anomalies in the pediatric population. although renal fibrosis and inflammation are involved in the pathophysiology of severe upj obstruction, the molecular mechanisms underlying evolution of the lesions remain undetermined. in addition, most of this information has been obtained in animal models of disease. recently discovered small non-coding rnas called mirnas are excreted and are stable in urine. hence, urinary mirnas could help to further decipher the pathophysiology of upj obstruction and generate the missing link between human and animal data. in this study we combined human urinary mirna data and animal kidney tissue mirna and mrna data in a systems biology approach to obtain insight in the pathophysiology of the disease and improve upon the translatability of the results. the combined approach and independent validation pointed to the consistent dysregulation of specific mirnas and to new potential targets in upj obstruction. mirnas let-7a-5p, mir-125b-5p, mir-16-5p, mir-26a-5p and mir-29b-3p were consistently modified in mice and humans. among these, mir-29b is a well-known player in renal pathologies and especially fibrosis. indeed, mir-29b targets specific fibrotic molecules including collagens or α-smooth muscle actin, and its abundance is reduced in many fibrotic pathologies as its expression is inhibited by tgfβ. the protective role of mir-29b in fibrosis was further demonstrated in vivo since restoring mir-29b levels in a diabetic nephropathy animal model reversed accumulation of renal extracellular matrix. on the other hand, to our knowledge, this is the first time that mir- and let-7a are associated to the development of kidney disease, as these mirnas were mostly characterized to be involved in cancer. downregulation of let-7a has also been observed in scleroderma, contributing to the excessive deposition of collagen and tissue fibrosis in the skin. few data is available for mir-125b and kidney disease. circulating mir-125b was found downregulated in chronic kidney disease in hypertensive patients suffering from cardiovascular disease. in another study upregulation of mir-125b protected against cisplatin-induced kidney injury via inhibition of nuclear factor erythroid-2-related factor. mir-26a has previously been reported to be over expressed in lung epithelia during development and to be involved in glomerular and tubular injury related to podocyte damage and maintenance of glomerular filtration rate. a recent small-scale study identified in kidney tissue five mirnas associated to upj obstruction. none of those overlapped with the human urinary mirnas in our study and one, mir-342-5p, was also found in kidney tissue to be associated to obstruction in the partial neonatal mouse model, however with an opposite regulation. a total of putative mrna targets for these differentially expressed mirnas were predicted with the use of ingenuity pathway analysis. in vitro validation of the predicted mirna-mrna pairs and in vivo assessment of the observed mrna changes led to the sound confirmation of the regulation of e ubiquitin-protein ligase dtx by let-7a. although e ubiquitin-protein ligase dtx has not yet been found associated to obstructive nephropathy, using the kidney & urinary pathway knowledge base we observed that it has been found to be induced in models of polycystic kidney disease and in a lupus nephritis model. furthermore, dtx is a member of notch signaling non-canonical pathway. it has been described that activation of notch signaling can lead to fibrosis. hence one can hypothesize that downregulation of let-7a, activates dtx and the notch signaling pathway, promoting the progression of fibrosis in upj obstruction. nav is a protein mostly found in neurons and is reported to play a critical role in microtubule development, but has not yet been implicated in upj obstruction. coinciding with dtx nav expression is also induced in a model of polycystic kidney disease and is induced in vitro by the major pro-fibrotic cytokine tgfβ, potentially linking nav induction to fibrosis. it is notable, though, that the regulation of nav by mir-29b-3p did not follow the classical regulation pattern in the partial uuo model. in contrast, in the in vitro experiment nav followed the predicted regulation and confirming that it may be a direct target of mir-29b. previous reports have documented that some mirnas may not target mrnas directly, but only block the protein translation, leaving the mrna intact. furthermore, other reports point to the fact that mirnas may induce the same direction of regulation of their mrna targets depending on the timeframe and conditions, providing an explanation of a possible indirect mechanism of mir-29b-nav regulation. nevertheless, further investigation is needed to determine if nav is a direct target of mir-29b in vivo and if it is an interesting molecule in the context of upj obstruction. combination of the human urine mirna data with the mouse tissue data increased the confidence in the human data and allowed selecting the most promising mirnas in human disease. a downside of our approach is that this type of prioritization will only focus on part of the molecular mechanisms of upj. if signals for specific mechanisms are absent in, for example, humans due to technical limitations or absence of shedding of mirnas in urine but not in mouse tissue, this mechanism is not necessarily irrelevant in the pathophysiology of upj. another shortcoming of our study is the fact that we used unadjusted p-values. infants with upj obstruction included in the study displayed a wide range of hydronephrosis and pelvic diameter which generates considerable variability in the upj obstruction group. the mean fold change after comparison of healthy controls and upj obstruction patients was only fold in this data. this is probably due to the fact that during excretion/shedding of mirnas in urine the in-situ mirna changes are flattened out. moreover, correction for multiple testing resulted in no significant mirnas in humans. we therefore continued the prioritization with unadjusted p values but we compensated for this shortcoming by the independent validation of the selected targets. finally, the question remains whether observations in mouse tissue or human urine are faithfully reflecting the changes in the human kidney. in the past, studies have used a similar approach as ours, i.e. by combining urine and tissue analysis, and/or by combining human and animal data, to understand the role of mirnas in the development of renal lesions. for example, mir- has been found to be consistently increased in animal and human samples of both urine and kidney tissue in the context of renal fibrosis. however, during acute kidney injury, mir- was increased in kidney tissue and decreased in the urine in a rat model, and increased in human urine. in a study of wang et al. again in a kidney injury model, mir-10a and mir-30d were found decreased in tissue and increased in urine after injury. finally, mir- was observed in different studies to be up-regulated in tissue in animal model of fibrosis, but down-regulated in human tissue samples and increased in human urine samples. these examples show that there is no common regulation pattern for mirnas in urine and kidney and in different species, even in the same pathological context and despite the fact that their role in renal lesions has been established with confidence. but by taking advantage of the conservation ability of mirnas, combination of urine and tissue analysis, and/or human and animal data allows to identify features that are consistently and significantly modified as being associated with the disease with more confidence and can help transfer observations from animal models to human research. in our study, we did not observe enriched canonical pathways when studying the mirnas of human or mouse upj obstruction separately even if around hundreds of mirnas were found significantly different. hence we have used both statistical selection, combination of different omics data and pathway enrichment analysis to identify mirnas and their targets most likely involved in the etiology of upj obstruction.  collectively this 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 dtx in both man and mice that would not be identified otherwise. we believe that our approach of combining omics data is generally applicable. many omics studies generate long lists of differentially expressed molecules that are difficult to prioritize and does not necessarily inform on the actual impact of these changes in disease. to further improve on the validity and clinical translatability of the data, and because urine is a rich source of biomarkers that can be collected easily and non-invasively, combination of human urine and animal tissue data could be of great help to better understand the molecular mechanisms involved in the development of many complex diseases. 
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 dtx in both man and mice that would not be identified otherwise. in an attempt towards improved understanding of obstructive nephropathy and improved translatability of the results to clinical practice we have developed a systems biology approach combining omics data of both human and mouse obstructive nephropathy. although renal fibrosis and inflammation have shown to be involved in the pathophysiology of obstructive nephropathies, molecular mechanisms underlying evolution of these processes remain undetermined. in vitro and in vivo validation identified consistent dysregulation of let-7a-5p and mir-29-3p and new potential targets, e ubiquitin-protein ligase and neuron navigator, potentially involved in fibrotic processes, in obstructive nephropathy in both human and mice that would not be identified otherwise. obstructive nephropathymirnas/micrornasmicroarrayslet-7a-5p and mir-29b-3pdtx and nav1http://dx.doi.org/ /501100004963seventh framework programmepeople-itn-ga-2013–608332papadopoulos theofilos programme hospitalier de recherche cliniquercb 2007-a00854-49decramer stéphane fondation du rein sous égide de la fondation pour la recherche médicale et ses partenaires genzyme fdr-sdn/frmklein julie issue-copyright-statement© the author 2017 we have studied in parallel the urinary mirnome of infants with ureteropelvic junction obstruction and the kidney tissue mirnome and transcriptome of the corresponding neonatal partial unilateral ureteral obstruction mouse model.
 although renal fibrosis and inflammation have shown to be involved in the pathophysiology of obstructive nephropathies, molecular mechanisms underlying evolution of these processes remain undetermined. in an attempt towards improved understanding of obstructive nephropathy and improved translatability of the results to clinical practice we have developed a systems biology approach combining omics data of both human and mouse obstructive nephropathy.  we have studied in parallel the urinary mirnome of infants with ureteropelvic junction obstruction and the kidney tissue mirnome and transcriptome of the corresponding neonatal partial unilateral ureteral obstruction mouse model. several hundreds of mirnas and mrnas displayed changed abundance during disease. combination of mirnas in both species and associated mrnas let to the prioritization of five mirnas and 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, e ubiquitin-protein ligase and neuron navigator, potentially involved in fibrotic processes, in obstructive nephropathy in both human and mice that would not be identified otherwise.  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 dtx in both man and mice that would not be identified otherwise. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords obstructive nephropathymirnas/micrornasmicroarrayslet-7a-5p and mir-29b-3pdtx and nav1http://dx.doi.org/ /501100004963seventh framework programmepeople-itn-ga-2013–608332papadopoulos theofilos programme hospitalier de recherche cliniquercb 2007-a00854-49decramer stéphane fondation du rein sous égide de la fondation pour la recherche médicale et ses partenaires genzyme fdr-sdn/frmklein julie issue-copyright-statement© the author 2017 