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

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.

Diminished met signaling in podocytes contributes to the development of podocytopenia in transplant glomerulopathy.

Transplant glomerulopathy (TxG) can show secondary focal and segmental glomerulosclerosis (FSGS). FSGS in native kidneys is caused by podocytopenia. This study examines podocytopenia and the role of decreased paracrine Met activation on podocytes by decreased glomerular hepatocyte growth factor (HGF) levels in the development of podocytopenia in TxG. Podocytes were counted in 10 zero-hour biopsies and 10 specimens each with and without TxG. HGF/Met was examined with immunostains and quantitative RT-PCR in a set of three consecutive biopsies from 10 patients with TxG, including the diagnostic biopsy (DiagnBx) and the two previous biopsies (1stPrevBx and 2ndPrevBx). Antiapoptotic effects of HGF on podocytes were examined in vitro. Mean podocyte numbers per glomerulus were lower and glomerular volume higher in TxG. Fewer of the two preceding biopsies of the patients than of the controls contained phospho-Met(Tyr1349)-positive podocytes (2 of 8 versus 7 of 7, P = 0.0070; 4 of 9 versus 9 of 9, P = 0.0294). Glomerular HGF mRNA levels were lower in the 1stPrevBx of the patients (0.049 ± 0.083 versus 0.284 ± 0.331; P = 0.0155). In vitro, HGF stimulation of podocytes resulted in antiapoptotic phosphorylation of AKT and extracellular signal-regulated kinase (ERK) and induction of X-linked inhibitor of apoptosis protein (XIAP). Decreased antiapoptotic Met signaling in podocytes, probably due to decreased HGF secretion by glomerular epithelial cells, could contribute to podocyte loss and FSGS in TxG.

ADAMTS13--marker of contractile phenotype of arterial smooth muscle cells lost in benign nephrosclerosis.

BACKGROUND: Hypertensive nephrosclerosis alone and in combination with other renal diseases is a leading cause of terminal renal insufficiency. Histologic lesions manifest as benign nephrosclerosis (bN) with arteriolar hyalinosis and later fibrosis. Procoagulant micromilieus have been implicated in fibrosis. Hyalinosis is considered to consist of plasma insudation possibly containing procoagulant factors like von Willebrand factor (VWF). Therefore, it is hypothesized that VWF cleaving protease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type-1 motif, 13) is normally expressed by arteriolar vascular smooth muscle cells (VSMCs) and diminished in bN and that this reduction contributes to fibrosis in bN. METHODS: ADAMTS13 expression was examined by immunohistochemistry and quantitative real-time polymerase chain reaction in VSMCs of various human organs. Fifty-four specimens with and seven without bN were immunostained for ADAMTS13, VWF, CD61 and VSMC differentiation markers in arteriolar walls. RESULTS: Expression of ADAMTS13 is confirmed in VSMCs. In bN, ADAMTS13 immunostaining of arterial VSMCs correlated inversely with fibrotic but not hyalinotic lesions. Smooth muscle myosin heavy chain showed an inverse correlation with hyalinotic, as opposed to fibrotic lesions of bN. Smoothelin showed an inverse correlation with both hyalinotic and fibrotic lesions of bN. VWF was absent in normal controls and hyalinotic lesions, but present exclusively in fibrotic lesions in 7/54 (13%) bN cases. CD61 was absent in all arteriolar walls. CONCLUSIONS: The present results establish ADAMTS13 as a novel marker of contractile VSMCs that is retained in early hyalinotic bN but partially lost later in fibrotic bN. Loss of ADAMTS13 and accumulation of VWF in fibrotic but not hyalinotic arteriolar walls could further propagate fibrosis in bN.

Urinary excretion of IGFBP-1 and -3 correlates with disease activity and differentiates focal segmental glomerulosclerosis and minimal change disease.

The glomerular microenvironment is influenced by circulating growth factors that are filtered from the blood stream and pass the glomerular filtration barrier. In this study, we wanted to explore the role of IGF-binding proteins (IGFBPs) in two diseases that concern podocytes. We analyzed glomerular expression and urinary excretion of IGFBP-1, -2, and -3 in patients with focal segmental glomerulosclerosis (FSGS) or minimal change disease (MCD). We found that patients with active FSGS excrete high amounts of podocalyxin positive cells as well as IGFBP-1 and -3. In human podocytes, we can induce mRNA expression of IGFBP-3 in response to TGF-beta and in human microvascular endothelial cells expression of IGFBP-1 and -3 in response to TGF-beta and Bradykinin. We conclude that the local expression of IGFBPs in podocytes and endothelial cells might contribute to the pathogenesis of glomerular disease and that IGFBP-1 and -3 are potential non-invasive markers of FSGS.