Received date: June 21, 2017; Accepted date: July 26, 2017; Published date: July 31, 2017
Citation: Müller-Deile J, Dannenberg J, Liu P, Thum T, Lorenzen J, et al. (2017) Identification of Cell-and Disease-Specific Micro RNAs in Glomerular Pathologies . J Mol Genet Med 11: 277 doi:10.4172/1747-0862.1000277
Copyright: © 2017 Müller-Deile J, et al . This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
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Background: MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression in physiological processes as well as in diseases. Currently miRs are already used to find novel mechanisms involved in diseases and in the future, they might serve as diagnostic markers. To identify miRs that play a role in glomerular diseases urinary miR-screenings are a frequently used tool. However, miRs that are detected in the urine might simply be filtered from the blood stream and could have been produced anywhere in the body, so they might be completely unrelated to the diseases. Methods: We performed a combined miR-screening in pooled urine samples from patients with different glomerular diseases as well as in cultured human podocytes, human mesangial cells, human glomerular endothelial cells, and human tubular cells. The miR-screening in renal cells was done in untreated conditions and after stimulation with TGF-β. Results: A merge of the detected regulated miRs led us to identify disease-specific, cell type-specific and cell stress-induced miRs. Further, urinary miRs from patients with different glomerular diseases could be assigned to the different renal cell types. Conclusion: Thus, a combined urinary and cell miR-screening can improve the interpretation of screening results. These data are useful to identify novel miRs potentially involved in glomerular diseases.
MicroRNA-screening; Diagnostic marker; Glomerular diseases; Urinary micrornas; Cell type-specific microRNAs; Disease-specific microRNAs
Abbreviations: miR: microRNA; DN: Diabetic Nephropathy; FSGS: Focal Segmental Glomerulosclerosis; HUS: Haemolytic Uraemic Syndrome; IgA-GN: IgA-Glomerulonephritis; MCD: Minimal Change Disease; MGN: Membranous Glomerulonephritis; MPGN: Membrano- Proliferative Glomerulo-Nephritis; PREEC: Preeclampsia
In the nucleus primary miRs are transcribed by RNA polymerases and then cleaved into double-stranded miR precursors (Pre-miRs) by RNase III enzyme (Drosha) and DiGeorge syndrome critical region 8 (DGCR8) [1,2]. Pre-miRs are exported into the cytoplasm where they are further cleaved into a guide strand and a passenger strand by the enzyme Dicer. Then the guide strand is loaded onto the RISC that binds to the 3′ untranslated region (3′ UTR) of a target messenger RNA and inhibits RNA translation . Several miRs are enriched in human kidney and miRs seem to play a role in the glomerular homeostasis . Mice with podocyte-specific alteration in miR-expression by deletion of Dicer or Drosha display progressive glomerular damage with proteinuria and podocyte defects [6,7].
MiRs can be secreted in body fluids and therefore could possibly serve as biomarkers in various glomerular diseases. For example, patients with focal glomerulosclerosis (FSGS) had 10-times elevated concentrations of miR-3d and miR-10a in the urine compared to healthy controls . Urinary expression of miR-200a, miR-200b and miR-429 were down regulated in patients with IgA glomerulonephritis (IgA-GN) and this downregulation correlated with severity of the disease and rate of progression .
MiRs offer some important advantages over other markers as they are stable and protected from endogenous RNase because of their small size and by packaging within exosomes [10,11]. However, though suggested as potential biomarkers the origin of miRs has rarely been defined and cell type specific miRs have not yet been reported in the kidney.
In the past miR-screenings in body fluids or tissue samples of patients with glomerular diseases were compared to healthy controls [12-14]. By this approach, only comparing samples of one disease to control samples makes the specificity of the findings elusive. In addition, it is not clear if the miRs are “bystanders” or might originate from cells and tissues involved in the disease. Urinary miRs might be filtered from the blood, excreted by tubular cells or derived directly from glomerular cells affected by the disease process.
In this study, we described the advantages of a combined miR-screening in urine as well as in cultured renal cells and explained different ways of data normalisation and interpretation. We identified renal cell type-specific miRs and miRs specifically regulated by TGF-β in these cells. With this approach, we were able to investigate miRs in urines from patients with different glomerular diseases and could compare them to controls. In addition, we could assign the urinary miRs to the different renal cell types.
Under permissive conditions at 33°C, podocytes proliferate. When cultured at 37°C, the SV40 T-antigen was inactivated for cell differentiation. Culture medium for human podocytes was RPMI 1640 Medium (Roth, Karlsruhe, Germany) with 10% fetal calf serum (FCS; PAA Laboratories, Pasching, Australia), 1% Penicillin/ Streptomycin and 0.1% Insulin. Human proximal tubular cells were cultured with renal epithelial cell media (Promocell, Baden-Württemberg, Germany) with 5% FCS (PAA Laboratories, Pasching, Australia), 10 ng/ml recombinant human epidermal growth factor, 5 µg/ml recombinant human Insulin, 0.5 µg/ml Epinephrine, 36 ng/ml Hydrocortisone, 5 µg/ ml Transferrin and 4 pg/ml Triiodo-L-thyronine. Human glomerular endothelial cells (Clonetech, Mountain View, CA) were cultured in endothelial cell basal media (EBM™-2; CC-3156, Lonza; Fisher). This medium was added with endothelial cell growth medium that contains 0.1% hEGF, 0.1% hydrocortison, 0.4% hFGF-b, 0.1% VEGF, 0.1% R3-IGF-1, 0.1% Ascorbic Acid, 0.1% Heparin, 2% FBS and 0.1% GA. Human mesangial cells were cultured in Dulbecco’s Modified Eagles’s medium supplemented with 10% FCS and 1% Penicillin-Streptomycin. Culture Conditions were 37°C and 5% CO2 air atmosphere.
Cells were stimulated with 5 ng/ml human TGF-β1 (Sigma Aldrich, Darmstadt, Germany) or high glucose (50 nM). Cells were harvested 48h later with Quiazol for RNA isolation.
Urine sample preparation for miR-screening
Morning urine was collected from healthy volunteers and from patients with biopsy-proven glomerular diseases: focal segmental glomerulosclerosis (FSGS), membranous glomerulonephritis (MGN), membranoproliferative glomerulonephritis (MPGN), diabetic nephropathy (DN), minimal change disease (MCD), preeclampsia (PREEC), haemolytic uraemic syndrome (HUS) and IgA-glomerulonephritis (IgA-GN). Ethical approval was obtained from Ethics Committee of the Hanover Medical School (#1709-2013). In total 36 patients were included in the study. Urine samples (50 ml) were centrifuged at 15000 rpm for 15 min to pellet the cells and cellular debris. The cell-free urine supernatant was stored at -80°C until miR-screening analysis. Pooled urine samples from four patients per disease were used in the miR-screening.
MiR-isolation and miR-screening
Purification of total RNAs including miRs from cultured renal cells and cell-free urine from patients was done with miRNeasy Kit (QIAGEN, Venlo, Netherlands). QIAzol lysis reagent was added to the samples, mixed and incubated for 5 min. 5 µl of 5 nM Syn-cel-miR-39 was added to each urinary sample to control for variations during preparation and later normalisation for endogenous miRs. Chloroform was added to the samples, they were centrifuged and the upper phase containing RNA was transferred to a new collection tube. The RNA was then isolated with the help of RNeasy Mini spin columns and different buffers according to the manufactures’ instructions.
MiR-screening in all cell types and in all urine samples was done with TaqMan® Array Human MicroRNA Card Set v3.0 and Megaplex™ RT Primers Human Pool Set v3.0 (life technologis, Carlsbad, CA). The set enables quantitation of 754 human miRs and includes endogenous control for data normalization and one TaqMan® MicroRNA Assay not related to human as a negative control. Preamplification of miRs before the screening analysis was done with Megaplex™ PreAmp Primers, Human Pool Set v3.0 (life technologis, Carlsbad, CA) according to manufactures’ instructions.
We used the delta-delta cycle threshold (CT) method to normalise our miR-screening data and to generate fold changes in miR-expression after TGF-β stimulation and fold changes in miR-expression in urine samples from patients with glomerular diseases compared to control.
Delta-delta CT = delta CT (sample) – delta CT (reference) with delta CT (sample) = CT value for sample normalized to endogenous housekeeping gene and delta CT (reference) = CT value for calibrator normalized to endogenous housekeeping gene. The CT is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e., exceeds background level). CT levels are inversely proportional to the amount of target nucleic acid in the sample. The maximum allowable CT value in our study was set to 35. MiR-samples with bad intensity quality were excluded in the analysis. The normalisation of the miR-screening data of the different cell lines was done with the housekeeper U6 snRNA-001973.
Other like RNU48 or RNU44 was not used because they were regulated in our cell-screening. This is in line with published data showing that these small-nucleolar RNAs like RNU44, RNU48, RNU43 and RNU6B commonly used for miR-normalisation are regulated in tumors . The housekeeper for the miR-analysis in urines was cel-39 that was spiked into pooled urines of each disease before miR-isolation. Normalized CT values were then transformed into relative quantity (RQ) value according to formula 2- (delta-delta CT). MiRs with RQ values > 1.5 were considered upregulation and < 0.5 were considered downregulation.
We performed a Q-PCR based miR-screening (TaqMan® Array Human MicroRNA Card Set v3.0) in cultured human podocytes, human glomerular endothelial cells, human mesangial cells and human proximal tubular cells in unstimulated conditions and after stimulation with TGF-β. The same miR-screening was performed in pooled urine samples from patients with focal segmental glomerulosclerosis (FSGS), membranous glomerulonephritis (MGN), membranoproliferative glomerulonephritis (MPGN), diabetic nephropathy (DN), minimal change disease (MCD), preeclampsia (PREEC), haemolytic uremic syndrome (HUS), IgA-glomerulonephritis (IgA-GN) and healthy controls. The screening enabled the detection of 754 different human miRs
A schematic illustration of the miR-screening is given in Figure 1 Patients’ characteristics are given in Table 1. Urine samples of two women and two men (except PREEC: urine samples of four women) with an active form of their disease were used for the miR-screening.
|Disease||Age [years]||Sex||Serum Creatinine[µmol/l]||UPC Ratio [mg/ml]|
Table 1: Characteristics of patients from the urinary miR-screening.
Renal cell type-specific miRs
By comparing the individual expression levels of each miR in the different cell types to the mean expression level of this miR in all cell types, we were able to detect cell type-specific miRs (Supplementary Table 1). Thereby, we could identify miR-143-3p as podocyte-specific miR that was highly expressed in this cell type (fold change 36 in human podocytes compared to 0.37 in human glomerular endothelial cells, 0.27 in human mesangial cells and 0.25 in human tubular cells). In previous experiments we have already documented the importance of miR-143-3p for podocyte function and ultrastructure . Examples for a glomerular endothelial cell and a mesangial cells-specific miRs are miR-126 and miR-206 respectively. MiR-9 was exclusively expressed in tubular cells.
We examined the overlap of miR-expression in the different cell types. 29 different miRs were only expressed in cultured human mesangial cells, 32 miRs were specific for human glomerular endothelial cells, 65 miRs were only detectable in human podocytes and 38 miRs were specific for proximal tubular cells (Table 2). 19 different miRs could be detected in all four renal cell types (Figure 2).
|Endothelial Cells||Mesangial Cells||Podocytes||Tubular Cells|
Table 2: Cell type-specific miRs in cultured human glomerular endothelial cell, mesangial cells, podocytes and tubular cells.
Next, we compared the expression level of each miR and cell type to the global mean of all 754 miRs from the screening to categorize them in rather high (fold change >10) or low (fold change <0.5,) expressed (Supplementary Table 2). This analysis revealed that miR-126, miR-126#, miR-531 and mir-346 were not only glomerular endothelial cell-specific but also highly expressed in this cell type (fold change >10 compared to the global mean of all miRs). MiR-106a# and miR-302a were highly expressed only in mesangial cells. Furthermore, miR-200b, miR-1225, miR-221, miR-1267 and miR-331were specific for podocytes and expressed more than 10-fold in this cell type compared to the global mean. 4 different miRs were rather high expressed and specific for tubular cells: miR-1305, miR-499-3p, let-7b and miR-454 (Supplementary Table 2).
A third way of analysis is comparing the retrospective mean miR-expression levels of all cell types to the global mean of miR-expression level of all miRs, Again, we looked for miRs that were only expressed more than 10-fold in one cell type. This method gave similar not overlapping results compared to the data analysis above. MiR-126, miR-581, miR-1274A and miR-126# were endothelial cell-specific whereas miR-106a#, miR-484 and let 7b were specific for mesangial cell. For podocytes, the same miRs as in the analysis above came up: miR-200b, miR-1225, miR-221, miR-1267 and miR-331. MiR-1305, miR-520b and miR-486 were only upregulated in tubular cells more than 10-fold (Supplementary Table 3).
MiRs regulated by TGF-β in cultured renal cells
To identify cell stress inducible miRs we compared miR-profiles from cultured renal cell lines before and after stimulation with TGF-β. We generated fold changes in miR-expression levels after TGF-β stimulation (Supplementary Table 4) and looked at the context cell type-specific up- or downregulation of miRs. (Figures 3A-3D) gives the number of miRs upregulated (fold change > 1.5, red), downregulated (fold change < 0.5, green) or unregulated (fold change > 0.5 and < 1.5, overlap) after stimulation with TGF-β in human mesangial cells (Figure 3A), human glomerular endothelial cells (Figure 3B), human podocytes (Figure 3C) and human tubular cells (Figure 3D). As well as the 10 top, upregulated and 10 top downregulated miRs after TGF-β stimulation. Of note, most miRs were downregulated following the stimulation with TGF-β in all cell types.
Upregulation of miR-expression after TGF-β stimulation was most prominent in cultured glomerular endothelial cells and podocytes. Moreover, upregulation of miRs after TGF-β was cell type-specific for most miRs. For example, miR-378a-3p was specifically upregulated in cultured human podocytes. The importance of miR-378a-3p for glomerular filter function and ultrastructure was previously described by us .
Interestingly, the regulation of some miRs was concordant for more than one cell type. For example, miR-199a-3p was upregulated in both mesangial cells and podocytes. MiR-1247 was upregulated not only in podocytes but also in tubular cells. MiR-1243, miR-1225-3p, miR-520D-3p and miR-520c-3p were induced after stimulation with TGF-β in glomerular endothelial cells as well as tubular cells. This suggests that these cell types regulate common pathways. What is also striking is that we found no miR significantly upregulated in more than two cell types, indicating that the miR-regulation response after TGF-β is clearly cell type specific.
MiRs in cell-free urines from patients with different glomerular diseases compared to control
Next, we examined in a screening experiment urinary miR-profiles from pooled urine samples from patients with different glomerular diseases and compared them to those from healthy controls (Supplementary Table 5). Interestingly, except for ANCA and IgA-GN, most miRs were higher expressed in disease urines compared to controls. (Figure 4) gives the number of miRs upregulated (fold change > 1.5, red), downregulated (fold change < 0.5, green) or unregulated (fold change > 0.5 and < 1.5, overlap) in MGN (Figure 4A), PREEC (Figure 4B), IgA-GN (Figure 4C), DN (Figure 4D), FSGS (Figure 4E), MCD (Figure 4F), ANCA (Figure 4G), and HUS (Figure 4H),
The 10 top upregulated and 10 top downregulated miRs in the urines from patients compared to healthy controls are depicted in the charts of (Figures 4A-4H), Interestingly, only one miR was upregulated in all disease urines compared to control. This was miR-155.
MiR-155 also caught our attention in the miR-screening in the cells. It was much higher expressed in glomerular endothelial cells compared to other cell types (fold change 9.0, Supplementary Table 1). It consistently was detected in high fold change in glomerular endothelial cells in the miR-expression analysis with the global mean of all miRs and with the local mean of all miRs (Supplementary Tables 2 and 3). After stimulation with TGF-β miR-155 was further upregulated in glomerular endothelial cells (fold change 5.4, Supplementary Table 4).
We were able to identify disease-specific miRs that were only detectable in one glomerular disease and not present in controls. These disease-specific miRs were let-7g for MGN, miR-99b# for PREEC, miR-603 for FSGS and miR-590 for ANCA.
Assigning urinary miRs to the different renal cell types
By combining the cell and urinary miR-screenings, we could assign the miRs found in the urines from patients with glomerular diseases to the miRs identified in TGF-β stressed cultured renal cells (Figures 5A-5H). The combination of cell and urine miR-screenings enabled us to identify cell type-specific miRs in the different glomerular diseases (Supplementary Table 4 and Figures 5A-5H).
It is estimated that 60% of the total human proteome is regulated by about 2000 known miRs . MiRs also seem to play an important role in gene regulation in disease processes. Therefore, miR-screenings are novel tools to find diagnostic markers or even therapeutic targets in glomerular diseases. In the past miR-screenings were predominantly performed in serum or urine from patients. However, by analysing miRs in body fluids only, the origin of the miRs as well as their pathophysiological role in disease remains elusive. Urinary miRs might be filtered or excreted by the kidney. Alternatively, they might be directly derived from renal cells during the disease process (Figure 6).
We performed three different miR-screenings with 745 different miRs: One in different renal cell types under normal culture condition, one in cultured renal cell types after stimulation with TGF-β and another in urine samples from patients with different glomerular diseases. These data are the basis for different ways of analysis depending on the biological question to be answered.
Most studies on urinary miRs used the urine sediment obtained after low-speed centrifugation. However, a large number of low-quality and degraded RNA was recently detected in the urinary cell pellet . Therefore, we decided to use the cell-free supernatants of pooled patient urines in our miR-screening. We first wanted to identify potentially cell type-specific miRs. We could identify miR-126 as one of the glomerular endothelial cell-specific miRs in our screening. Well in line with this finding, miR-126 was already described as an endothelial cell-specific miR that governs vascular integrity in other tissue [20-23].
MiR-143-3p was predominantly expressed in podocytes in our miR-screening. We have confirmed the importance of this miR for the maintenance of a functional glomerular filtration barrier in the zebrafish model . MiR-30 family members were also highly expressed in our cultured human podocytes. In line with this, a role of the miR-30 family in podocyte homeostasis was previously described in mice .
The comparison of cellular miR-profiles before and after stimulation with TGF-β, revealed cell stress-induced miRs. Among the top 10 miRs upregulated after TGF-β most were cell type-specific and none was regulated in more than two renal cell types. Some cell type-specific regulations were consistent with previous findings. For example, miR-143-3p and miR-378a-3p are known to be regulated by TGF-β in non-renal cells [25,26]. Both miRs were also upregulated in cultured human podocytes after stimulation with TGF-β in our miR-screening. Circulating miR-210 predicts survival in critically ill patients with acute kidney injury . Interestingly, miR-210 was upregulated after TGF-β in human mesangial cells in our study. MiR-199a-3p was upregulated in human mesangial cells and podocytes after stimulation with TGF-β. A known target of miR-199a-3p is versican also produced by podocytes and mesangial cells [28,29].
Regarding the urinary miR-screening most miRs were higher expressed in patients with glomerular diseases compared to healthy controls. This might be due to the increased leakiness of the glomerular filtration barrier in glomerular disease that also allows more microparticles and RNA-binding proteins associated with miRs to pass . Recently, urinary miR-21, miR-200c and miR-423 have been identified as sensitive indicators of kidney injury. MiR-21 was also described as positive miRs inhibiting pathophysiological pathways in DN . Nevertheless, the origin of these urinary miRs is unknown .In our screening, miR-21 was expressed in cultured human mesangial cells and podocytes and was detectable as upregulated in urines from patients with IgA-GN, FSGS, MCD, MGN, PREEC and DN. MiR-200c was upregulated in PREEC, DN, MGN, FSGS, cultured human mesangial cells and podocytes and miR-423 was detectable in all four renal cell types and in urines from patients with DN and MGN. MiR-378a-3p was found in urines samples from patients with MGN, FSGS and MCD and upregulated in cultured human podocytes in response to TGF-β treatment. In a previous study we could confirm the importance of miR-378a-3p for glomerular function as it is a regulator of the glomerular matrix protein nephronectin . Thus, our data confirm that urinary miRs seem to be markers for renal injury. Interestingly one miR was upregulated in all disease urines compared to control, this was miR-155.
In all different analysis strategies in cells including the analysis after TGF-β-stimulation, miR-155 was highly upregulated in glomerular endothelial cells. TGF-β-regulation of miR-155 has been described before . Well in line with our observation miR-155 was previously described by others to play an important role in endothelial cell activation [34,35].
Combining the cellular and urinary miR-screening, we were able to define disease-specific and cell type-specific miR-profiles and could assign the urinary miRs to the different renal cell types. To our knowledge this is the first study merging biological samples from patients with results from unstressed and stressed cultured cells to identify biological important miRs. Even though our screening to identify pathophysiologically relevant miRs confirmed several published previous observations by us and others, a limitation of our study is that the screening results have to be confirmed in independent experiments or larger patient cohorts. Nevertheless, our approach uses a unique comparison in eight different disease groups and all corresponding glomerular cell types and is therefore a first approach to identify novel miRs potentially involved in the pathophysiology of glomerular diseases.
All patients gave consent that their urine can be used for miR-analysis. The authors declare that they have no competing interests. JMD is supported by internal medical school funding programs (Hochschulinterne Leistungsförderung (Hilf) and Junge Akademie Program (JA-MHH) of Hannover Medical School).