PLoS One. 2016 Mar 1;11(3):e0150154.

Urinary Exosomal miRNA Signature in Type II Diabetic Nephropathy Patients.

Delić D1, Eisele C1, Schmid R1, Baum P1, Wiech F1, Gerl M1, Zimdahl H2, Pullen SS3, Urquhart R4.
  • 1Translational Medicine & Clinical Pharmacology, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorferstr.65, 88397 Biberach, Germany.
  • 2R&D Project Management, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorferstr.65, 88397 Biberach, Germany.
  • 3Cardiometabolic Diseases Research, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06877-0368, United States of America.
  • 4Translational Medicine & Clinical Pharmacology, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06877-0368, United States of America.

 

Abstract

MicroRNAs (miRNAs) are short non-coding RNA species which are important post-transcriptional regulators of gene expression and play an important role in the pathogenesis of diabetic nephropathy. miRNAs are present in urine in a remarkably stable form packaged in extracellular vesicles, predominantly exosomes. In the present study, urinary exosomal miRNA profiling was conducted in urinary exosomes obtained from 8 healthy controls (C), 8 patients with type II diabetes (T2D) and 8 patients with type II diabetic nephropathy (DN) using Agilent´s miRNA microarrays. In total, the expression of 16 miRNA species was deregulated (>2-fold) in DN patients compared to healthy donors and T2D patients: the expression of 14 miRNAs (miR-320c, miR-6068, miR-1234-5p, miR-6133, miR-4270, miR-4739, miR-371b-5p, miR-638, miR-572, miR-1227-5p, miR-6126, miR-1915-5p, miR-4778-5p and miR-2861) was up-regulated whereas the expression of 2 miRNAs (miR-30d-5p and miR-30e-5p) was down-regulated. Most of the deregulated miRNAs are involved in progression of renal diseases. Deregulation of urinary exosomal miRNAs occurred in micro-albuminuric DN patients but not in normo-albuminuric DN patients. We used qRT-PCR based analysis of the most strongly up-regulated miRNAs in urinary exosomes from DN patients, miRNAs miR-320c and miR-6068. The correlation of miRNA expression and micro-albuminuria levels could be replicated in a confirmation cohort. In conclusion, urinary exosomal miRNA content is altered in type II diabetic patients with DN. Deregulated miR-320c, which might have an impact on the TGF-β-signaling pathway via targeting thrombospondin 1 (TSP-1) shows promise as a novel candidate marker for disease progression in type II DN that should be evaluated in future studies.

PMID: 26930277

 

Supplement

Diabetic nephropathy (DN), the most frequent form of chronic kidney disease, is a progressive kidney disease and a major debilitating complication of both type 1 and type 2 diabetes that can lead to end-stage renal disease (ESRD) (1). Albuminuria is widely used as a biomarker for DN but recent studies suggest that micro-albuminuria is a less precise predictor of DN risk (2). Therefore, there is an unmet need to identify biomarkers reflecting early pathophysiological events during disease development and progression and which potentially allow monitoring predictive and/or prognostic treatment effects.

TGF-β is the key signaling pathway in DN pathogenesis which induces the synthesis and accumulation of extracellular matrix (ECM) leading to fibrosis and hypertrophy in renal cells under diabetic conditions (3). Several miRNAs are implicated to be functionally cross-linked with the TGF-β induced expression of ECM genes during the pathogenesis of DN. TGF-β responsive miRNA-regulatory networks were demonstrated in various kidney cells, such as proximal tubular epithelial cells, mesangial cells and podocytes. Up-regulated glomerular miR-21 expression is positively associated with albumin-creatinine ratio (ACR) in patients with DN and potentially serves as an indicator of podocyte damage (4). In contrast, miRNA members of the miR-200-, miR-30- and miR-29-families protect the kidney from fibrotic damage and their expression levels are decreased in various kidney cells types. In particular, the DPP-4 inhibitor linagliptin has been shown to inhibit TGF-β-induced endothelial to mesenchymal transition (EndMT) in kidneys of diabetic mice by restoring miRNA-29s level (5).

 

 

fig1Figure 1. Expression of differentially expressed TGF-β associated miRNAs in DN patients analyzed by microarrays. Fold induction of expression of up-regulated miR-21 (a) and down-regulated members of the miR-30 (b), miR-29 (c) and miR-200 (d) families in diabetic nephropathy patients (DN) and type 2 diabetic patients (T2D) was normalized to the mean expression of healthy controls (C). Significant differences to controls are indicated by * (P<0.05).

 

Urine contains various types of extracellular vesicles, exosomes being the most-well characterized so far which are secreted in large quantities from all nephron segments (6) and may thus provide valuable insights in renal pathophysiology. Recent studies revealed that urinary exosomal miRNA content is changed in patients with focal segmental glomerulosclerosis (7) and in type I diabetic patients with incipient diabetic nephropathy (8). Moreover, miR-29c level in urinary exosomes was shown to potentially serve as predictor of early fibrosis in lupus nephritis (9).

In the present study, using Agilent`s miRNA microarrays we identified an altered miRNA signature in urinary exosomes from type II DN patients for the first time. This deregulation of distinct urinary exosomal miRNA species correlates with degree of micro-albuminuria. Notably, the enriched expression of miRNA-320c which is indirectly involved in TGF-β signaling via targeting thrombospondin 1 (TSP-1) may represent a novel candidate marker for early progression of disease and/or early treatment effects. Furthermore, our study shows a clear trend to up-regulated miR-21 and down-regulated miR-200, miR-30 and miR-29 levels which, despite of miR-30d and miR-30e, did not pass our cut-off criteria (2-fold deregulation, adj. P-value<0.05) (Figure 1). The TGF-β responsive miRNA-regulatory networks in the different kidney cell types which are reflected in our data from urinary exosomal miRNA profiling in DN patients are summarized in Figure 2. Further studies are needed with larger cohorts characterized by a wider eGFR range showing a graded correlation of miRNA levels with UACR across the spectrum of normo- to micro- and macro-albuminuria. The importance of this pilot study is that it shows first insights into altered urinary exosomal miRNA profile in type II DN patients and that these novel, non-invasive markers show promise as tools for the mechanistic investigation of renal pathophysiology, including disease progression, and potentially allowing to monitor treatment effects.

 

 

fig2Figure 2. Schematic overview of TGF-β associated miRNA-regulatory networks in kidney cells which are reflected in urinary exosomes of type II DN patients. Up and down directed black arrows indicated up- and down-regulation, respectively. Amplification and inhibition of signaling is depicted in green and red, respectively.

 

References

(1)   Chan G, Tang SC. Current practices in the management of diabetic nephropathy. J R Coll Physicians Edinb. 2013; 43: 330–332.

(2)   Fried LF, Lewis J. Rebuttal of the Pro View: Albuminuria Is an Appropriate Therapeutic Target in Patients with CKD. Clin J Am Soc Nephrol. 2015; 10: 1095–1098.

(3)   Chen S, Jim B, Ziyadeh FN. Diabetic nephropathy and transforming growth factor-beta: transforming our view of glomerulosclerosis and fibrosis build-up. Semin Nephrol. 2003; 23: 532-43.

(4)   Lai JY, Luo J, O’Connor C, Jing X, Nair V, Ju W, et al. MicroRNA-21 in glomerular injury. J Am Soc Nephrol. 2015; 26: 805–816.

(5)   Kanasaki K, Shi S, Kanasaki M, He J, Nagai T, Nakamura Y, et al. Linagliptin-mediated DPP-4 inhibition ameliorates kidney fibrosis in streptozotocin-induced diabetic mice by inhibiting endothelial-to-mesenchymal transition in a therapeutic regimen. Diabetes. 2014; 63: 2120–2131.

(6)   Erdbrügger U, Le TH. Extracellular Vesicles in Renal Diseases: More than Novel Biomarkers? J Am Soc Nephrol. 2016; 27: 12–26.

(7)   Ramezani A, Devaney JM, Cohen S, Wing MR, Scott R, Knoblach S, et al. Circulating and urinary microRNA profile in focal segmental glomerulosclerosis: a pilot study. Eur J Clin Invest. 2015; 45: 394–404.

(8)   Barutta F, Tricarico M, Corbelli A, Annaratone L, Pinach S, Grimaldi S, et al. Urinary exosomal micro-RNAs in incipient diabetic nephropathy. PLoS One. 2013; 8: e73798.

(9)   Solé C, Cortés-Hernández J, Felip ML, Vidal M, Ordi-Ros J. miR-29c in urinary exosomes as predictor of early renal fibrosis in lupus nephritis. Nephrol Dial Transplant. 2015; 30: 1488–1496.

 

 

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