Abstract
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. 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. A merge of the detected regulated miRs led us to identify disease‐specific, cell type‐specific and cell stress‐induced miRs. Most miRs were down‐regulated following the stimulation with TGF‐β in all cell types. Up‐regulation of miRs after TGF‐β was cell type‐specific for most miRs. Furthermore, urinary miRs from patients with different glomerular diseases could be assigned to the different renal cell types. Most miRs were specifically regulated in one disease. Only miR‐155 was up‐regulated in all disease urines compared to control and therefore seems to be rather unspecific. In conclusion, 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.
Highlights
MicroRNAs are small non‐coding RNAs that play an import‐ ant role in gene regulation.[1,2,3] In the nucleus primary miRs are tran‐ scribed by Ribonucleic acid (RNA) polymerases and cleaved into double‐stranded miR precursors (Pre‐miRs) by RNase III en‐ zyme (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
The same miR‐screening was performed in pooled urine samples from patients with focal segmental glomerulosclerosis (FSGS), membranous glomerulonephritis (MGN), membranoproliferative glomerulone‐ phritis (MPGN), diabetic nephropathy (DN), minimal change dis‐ ease (MCD), PREEC, haemolytic uraemic syndrome (HUS), IgA glomerulonephritis (IgA‐GN) and healthy controls
Thereby, we could identify miR‐143‐3p as podocyte‐specific miR that was highly expressed in this cell type
Summary
MicroRNAs (miRs) are small non‐coding RNAs that play an import‐ ant role in gene regulation.[1,2,3] In the nucleus primary miRs are tran‐ scribed by Ribonucleic acid (RNA) polymerases and cleaved into double‐stranded miR precursors (Pre‐miRs) by RNase III en‐ zyme (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. We were able to investigate miRs in urines from patients with different glomerular diseases and could compare them to controls. We used the delta‐delta cycle threshold (CT) method to normalize 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.
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