In this paper we report the changes of urinary miRNA spectrum in T1D patients with different stages of albuminuria and nephropathy. We found concentration changes on specific miRNAs that may involve in specific pathways known to be altered in various forms of renal diseases. Since the kidney is the most likely source of these urinary miRNAs, we suggest that these miRNAs may be of biological and clinical significance in T1D.
A global Principal Component Analysis viewpoint of the microRNA profiles analyzed in this report suggests that there are some differences in the expression of urinary microRNA which appear to follow the clinical classification of patients and urinary samples with respect to albumin excretion. The apparent clustering of profiles from patients who had been matched into pairs, suggests that there are other factors affecting urinary microRNA besides the clinical classification of disease. Such factors are likely related to the variables we used in patient matching e.g. age, sex, and duration of disease and level of glycemic control. This observation justifies post-hoc our decision to explore specific microRNA signatures across the spectrum of clinical classification of patients and samples using a matched case control design.
Our matched case-control Bayesian analyses highlight a set of 27 differentially regulated miRNAs across different clinical stages of diabetic renal disease. Previous work using experimental, clinical chemistry or biopsy samples has demonstrated differential expression of many of these miRNAs in a variety of renal conditions: hypertensive nephrosclerosis
(with an increase of miR-429 levels in human renal biopsies 
), mouse models of chronic renal injury (increased miR-214 levels 
) and renal senescence
(increased miR-335 levels). Other miRNAs have also been implicated in immunologically mediated renal diseases such as lupus nephritis
, miR-638,miR-373-5p and miR-92b-5p 
), IgA nephropathy
(miR-429 correlating with the level of proteinuria and renal function 
), and acute T cell rejection of renal allografts
(decreased miR-323-5p, miR-638/miR-373-5p 
Based on miRNA target prediction databases, miRNAs showing concentration changes in diabetic urine may regulate genes that play key roles in renal physiology and pathophysiology: fibronectin
a key component of the extracellular matrix that accumulates in diabetic nephropathy 
which is also regulated in senescence models of renal proximal tubule epithelial cells 
responsible for polycystic kidney disease (miR-17-5p 
, superoxide dismutase, a mitochondrial antioxidant enzyme in renal mesangial cells(miR-335 
a key component of the tight junction in the thick ascending limb (has-miR-323b-5p 
), the tumor suppressor protein PTEN which is decreased in DN 
(and is directly regulated by miR-221-3p/222-3p 
in heterologous systems), Abcg2
(a stem cell marker 
regulated by miR-520h 
(a tumor suppressor gene involved in renal tumours targeted by miR-92a 
). Hence, prior research highlights a kidney related role for a number of the miRNAs found to be differentially expressed in our analyses, suggesting that these miRNAs may be important mediators of renal damage rather than simple biomarkers of an underlying injury process without pathobiological significance.
In addition, intriguing connections in heterologous systems have been reported for other miRNAs highlighted in this report: miR-221-3p/222-3p (neovascularization and vascular neointimal hyperplasia 
, Advanced Glycosylation End product mediated vascular damage 
), miR-424 (regulating angiogenesis in the setting of hypoxia by targeting Cul2
as well as Vegfr2
). Many of these conditions have been recognized as clinically important vascular complications of diabetes, often presenting simultaneously with the development of nephropathy; hence one may conjecture that the spectrum of urine miRNAs may allow one to stratify the risk of diabetic patients for developing extrarenal complications.
With the samples used in this study, we could not verify the association of miR-192 with DN. Higher miR-192 levels have been previously linked to renal damage in the streptozocin (T1D) and the db/db (T2D) mouse nephropathy models 
through TGFβ –mediated production of miR-192 by mesangial cells. More recent evidence points towards a positive feedback loop for TGFβ production involving miR-192 and miR-200b/c in mesangial cells 
. On the other hand, decreased
miR-192 was noted in biopsy specimens of patients with advanced diabetic nephropathy, while miR-192 expression was positively correlated with EGFR and negatively correlated with the degree of fibrosis suggesting a protective role for miR-192 
. In that report, miR-192 expression was predominantly localized to tubular epithelial cells and TGF exposure was found to decrease both miR-192 and E-cadherin mRNA levels. Hence it appears that miR-192 may be regulated differently in different renal cell populations, possibly in a DN stage specific manner. This hypothesis is supported by recent evidence which failed to detect alterations in miR-192 expression in microdissected glomeruli of Munich Wistar Fromter rat model of spontaneous develop diabetic nephropathy 
. Since the miRNAs in the urine originate from diverse cellular sources in the kidney, the lack of a differential expression of miR-192 in this report may reflect the cancelation of two diverging (positive in mesangial, negative in tubular epithelial cells) signals leading to an overall “null” effect.
Most of the identified miRNAs exhibited changes in one disease state rather than showing a quantitative trend of increasing or decreasing expression paralleling the severity of albuminuria. To understand this pattern we examined the predicted targets of these miRNAs and the corresponding pathways using structured vocabularies for biological annotation. Despite the disparate identity of the miRNAs, the mRNAs that are predicted to be targeted by them map to pathways that have been previously shown to be pathophysiologically relevant to DN: TGF (the prototypical “renal-fibrosis” culprit 
), PDGF (associated with mesangial proliferation and fibrosis 
) and FGF (clinical predictor of progression in diabetic nephropathy 
Our analyses suggest the involvement of NGF (Nerve Growth Factor, a prototypical Central Nervous System trophic molecule) in diabetic nephropathy. This may lead to a new direction toward the development of T1D associated nephropathy since so far the renal expression of NGF has been thought to reflect the level of glycemic control 
. Nevertheless, NGF has been recently shown to be involved in tissue repair and fibrosis in liver, skin and lung 
, and its involvement in non-diabetic renal disease has been noted in a number of biopsy studies over the last 30 years 
, so that the association of NGF with diabetic nephropathy appears plausible.
Growth Factor as well as other pathways (e.g. cell-cell and cell-matrix) are targeted from the microalbuminuric stage, while the number of targeted genes in these pathways increased at the overt nephropathy stage. Hence an “exposure-response” relation appears at the target (mRNA) rather than the regulator (miRNA) level. This relation stems from the overlapping, combinatorial, binding specificities of miRNAs to their mRNA targets so that the same pathways may be targeted by rather different sets of miRNAs depending on the prevailing cellular context.
An interesting aspect of the targets associated with the miRNAs identified in this study is the lack of an overwhelming association between growth factor transduction pathways and the tempo
of MA. Rather, an association with tissue damage, innate immunity, metabolic pathway and developmental program (re)-activation was shown, suggesting that recurrent bouts of metabolic or free oxidative stress may account for the persistency and possibly the progression of MA to overt nephropathy. To the extent that these statistically determined patterns are verified experimentally, further development of miRNA target identification may have potential clinical implications as an early diagnostic test for diabetic renal disease or to select and or monitor response to emerging therapies for diabetic renal disease; e.g. pentoxifylline 
, pirfenidone 
and bardoxolone 
which interfere with pathways implicated in our analyses.
The findings of our study should be interpreted in light of a number of limitations. First, we analyzed urine samples from an era in which current therapies for diabetic nephropathy (angiotensin converting enzyme inhibitors and angiotensin receptor blockers) were not widely used early in the disease process. Hence most of the patients with MA were not on ACEi/ARB inhibition even though evidence from randomized trials suggest that these agents delay the appearance of microalbuminuria 
. On the other hand, most patients with overt nephropathy were on such agents with persistence of their macroalbuminuric state. Hence, our findings reflect the natural urinary miRNA phenotype of the early stages of diabetic nephropathy, the failing treatment one in advance disease and are not proposed to be representative of patients undergoing optimal treatment with these agents. Although this would appear to represent a major limitation of this study, the data presented here are rather unique in that they provide information on both untreated patients as well as those failing therapy, allowing some insight into the pathways that underline treatment resistance to the current treatment paradigm. This is exemplified by miR-324-3p which was apparently increased in patients with PMA not receiving an ACEi in accordance with recent animal data suggesting that this miRNA is a promoter of renal fibrosis and is downregulated by ACEi inhibition. At the same time, our patients with overt nephropathy showed no tendency of this miRNA to change relative to controls (FC was 1.06 in this dataset) suggesting that some of the discordance in miRNA profiles may be the result of therapies preferentially affecting certain miRNA species but not others. Since this investigation never intended to delineate treatment induced changes in urine miRNA profiles, future studies should examine both responders and non-responders at different points in time to determine miRNA correlates of therapeutic success and failure. Second, while our experience is no different from previous studies examining urine miRNA profiles in renal transplantation 
, systemic lupus 
and chronic kidney disease 
, many of the urinary miRNA signals in this analysis were of low magnitude requiring a large number of PCR cycles and careful optimization of qPCR conditions 
to be detected. Third, we inferred the renal origin of urine miRNAs yet the possibility that the latter derive from other sources such as plasma cannot be ruled out. As the approximate molecular weight of miRNAs (~6.2–7.2 kDa) is below the permselectivity threshold of the glomerular filtration barrier (~ 60 kDa) it is possible that a substantial portion of circulating plasma miRNAs is ultrafiltered in the urine. Nevertheless, a recent study in chronic kidney disease found a dissociation between plasma and urine miRNA spectrum 
suggesting a substantial non-plasma source for urine miRNA. To resolve these issues, simultaneous profiling of plasma and urine should be undertaken, a task which was not possible in this report due to the unavailability of plasma samples. Fourth, some of the miRNAs identified as differentially regulated have been found to play a role in non-diabetic renal disease, so that the reported associations may lack disease specificity. We tried to overcome this limitation by combining the changes in miRNA concentrations with the simultaneous predictions of miRNA targets. Most of the pathways identified have been linked to the development of diabetic nephropathy among different animal models and clinical studies which suggests the combination of using specific miRNA levels and its interacting mRNA targets as a general approach to enhance interpretability and specificity of miRNA profiles. Furthermore, the use of panels of markers will be much more informative and can potentially distinguish pathologies that produce overlapping sets of markers.
In summary, a set of 27 differentially miRNAs were identified in matched urine samples from T1D patients with different stages of diabetic nephropathy, whose renal outcomes had been ascertained after prolonged follow up. These miRNAs map to pathways of known relevance to the development of diabetic renal disease, strongly suggesting the renal source of the miRNAs. Our results suggest that a number of miRNAs in urine may serve not only as molecular signatures of distinct clinical phenotypes in diabetic nephropathy but also as early indicators of alterations in specific biological processes in the kidney which can be of importance in individualizing emergent therapies for diabetic kidney disease. Further studies are needed to extend these observations in the setting of T2D and clarify the potential utility of these miRNAs in early diagnosis, risk stratification for progression and treatment selection or monitoring.