Dataset Information

Comparison of different normalization strategies for the analysis of glomerular microRNAs in IgA nephropathy.

ABSTRACT: Small nucleolar RNAs (snoRNAs) have been used for normalization in glomerular microRNA (miRNA) quantification without confirmation of validity. Our aim was to identify glomerular reference miRNAs in IgA nephropathy. We compared miRNAs in human paraffin-embedded renal biopsies from patients with cellular-crescentic IgA-GN (n?=?5; crescentic IgA-GN) and non-crescentic IgA-GN (n?=?5; IgA-GN) to mild interstitial nephritis without glomerular abnormalities (controls, n?=?5). Laser-microdissected glomeruli were used for expression profiling of 762 miRNAs by low-density TaqMan arrays (cards A and B). The comparison of different normalization methods (GeNormPlus, NormFinder, global mean and snoRNAs) in crescentic IgA-GN, IgA-GN and controls yielded similar results. However, levels of significance and the range of relative expression differed. In median, two normalization methods demonstrated similar results. GeNormPlus and NormFinder gave different top ranked reference miRNAs. Stability ranking for snoRNAs varied between cards A and B. In conclusion, we suggest the geometric mean of the most stable reference miRNAs found in GeNormPlus (miR-26b-5p), NormFinder (miR-28-5p) and snoRNAs (RNU44) as reference. It should be considered that significant differences could be missed using one particular normalization method. As a starting point for glomerular miRNA studies in IgA nephropathy we provide a library of miRNAs.

SUBMITTER: Bockmeyer CL

PROVIDER: S-EPMC4995590 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Comparison of different normalization strategies for the analysis of glomerular microRNAs in IgA nephropathy.

Bockmeyer Clemens L CL Säuberlich Karen K Wittig Juliane J Eßer Marc M Roeder Sebastian S SS Vester Udo U Hoyer Peter F PF Agustian Putri A PA Zeuschner Philip P Amann Kerstin K Daniel Christoph C Becker Jan U JU

Scientific reports 20160824

Small nucleolar RNAs (snoRNAs) have been used for normalization in glomerular microRNA (miRNA) quantification without confirmation of validity. Our aim was to identify glomerular reference miRNAs in IgA nephropathy. We compared miRNAs in human paraffin-embedded renal biopsies from patients with cellular-crescentic IgA-GN (n = 5; crescentic IgA-GN) and non-crescentic IgA-GN (n = 5; IgA-GN) to mild interstitial nephritis without glomerular abnormalities (controls, n = 5). Laser-microdissected glom ...[more]

PMID: 27553688

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Project description:Background: In a previous study of patients with assumed benign IgA nephropathy (IgAN), we have shown that 18.6% develop a progressive clinical course, which was not predicted by any known clinical nor histopathological parameters (Knoop 2017). We aim to differentiate between future progressors and non-progressors with assumed benign IgAN by using glomerular transcriptomics from diagnostic kidney biopsies and to investigate whether this can identify drugs to be used in those patients. Methods: We included adult progressors (n=15) and patients with clinical remission (non-progressors, n=21) from our previously reported IgAN cohort. Glomerular cross-sections were obtained through laser-capture microdissection from 10µm archival kidney biopsy sections for mRNA sequencing, using the SMARTer Stranded Total RNA-Seq kit – Pico Input Mammalian (Clontech Laboratories, California, USA). Key results were validated in external cohorts and on the protein level with immunohistochemistry in the discovery cohort and an internal validation cohort. We also compared our transcriptomic findings with an external genome-wide association study (GWAS) dataset (Kiryluk 2021). Results: For the statistical analyses, we studied 8 progressors and 9 non-progressors. Based on the 1,240 differentially expressed glomerular genes from the initial kidney biopsies, we were able to identify future IgAN progressors and non-progressors using a two-component classifier. The classifier predicted disease progression with 88% accuracy, 75% sensitivity and 100% specificity (AUC=0.875), more accurate than the recommended International IgAN Prediction Tool in this specific patient cohort. Among our differentially expressed genes, 10 were also found in the GWAS dataset (Kiryluk 2021). We identified 45 drug targets in the DrugBank database, including angiotensinogen, a target of sparsentan, currently investigated as therapy in IgAN (NCT03762850 and NCT04663204). The findings on upregulation of angiotensinogen, which was independent of blood pressure, were validated in an internal IgAN validation cohort. Interestingly, complement activation did not play a major role in our data. Our work suggests that LXR, FXR and macrophage activation pathways could be critically involved in the inhibition of the progression of low-risk ccRCC. Furthermore, a 10-component classifier could support an early identification of apparently low-risk ccRCC patients.

Project description:BackgroundIgA nephropathy (IgAN) and IgA vasculitis with nephritis (IgAVN) are related glomerular diseases characterized by marked similarities in immunological and histological findings. We herein performed a comparative proteomic analysis of glomerular proteins in IgAN and IgAVN.MethodsWe used renal biopsy specimens from 6 IgAN patients without nephrotic syndrome (NS) (IgAN-I subgroup), 6 IgAN patients with NS (IgAN-II subgroup), 6 IgAVN patients with 0-8.0% of glomeruli with crescent formation (IgAVN-I subgroup), 6 IgAVN patients with 21.2-44.8% of glomeruli with crescent formation (IgAVN-II subgroup), 9 IgAVN patients without NS (IgAVN-III subgroup), 3 IgAVN patients with NS (IgAN-IV subgroup), and 5 control cases. Proteins were extracted from laser microdissected glomeruli and analyzed using mass spectrometry. The relative abundance of proteins was compared between groups. An immunohistochemical validation study was also performed.ResultsMore than 850 proteins with high confidence were identified. A principal component analysis revealed a clear separation between IgAN and IgAVN patients and control cases. In further analyses, 546 proteins that were matched with ≥ 2 peptides were selected. The levels of immunoglobulins (IgA, IgG, and IgM), complements (C3, C4A, C5, and C9), complement factor H-related proteins (CFHR) 1 and 5, vitronectin, fibrinogen chains, and transforming growth factor-β inducible gene-h3 were higher (> 2.6 fold) in the IgAN and IgAVN subgroups than in the control group, whereas hornerin levels were lower (< 0.3 fold). Furthermore, C9 and CFHR1 levels were significantly higher in the IgAN group than in the IgAVN group. The abundance of some podocyte-associated proteins and glomerular basement membrane (GBM) proteins was significantly less in the IgAN-II subgroup than in the IgAN-I subgroup as well as in the IgAVN-IV subgroup than in the IgAVN-III subgroup. Among the IgAN and IgAVN subgroups, talin 1 was not detected in the IgAN-II subgroup. This result was supported by immunohistochemical findings.ConclusionsThe present results suggest shared molecular mechanisms for glomerular injury in IgAN and IgAVN, except for enhanced glomerular complement activation in IgAN. Differences in the protein abundance of podocyte-associated and GBM proteins between IgAN and IgAVN patients with and without NS may be associated with the severity of proteinuria.

Dataset Information

Comparison of different normalization strategies for the analysis of glomerular microRNAs in IgA nephropathy.

Publications

Comparison of different normalization strategies for the analysis of glomerular microRNAs in IgA nephropathy.

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