Putri Andina Agustian

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-β and in human microvascular endothelial cells expression of IGFBP-1 and -3 in response to TGF-β 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.

Glomerular mRNA expression of prothrombotic and antithrombotic factors in renal transplants with thrombotic microangiopathy.

Background Thrombotic microangiopathy (TMA) in renal transplants (rTx-TMA) is a serious complication and is usually either recurrent TMA (RecTMA) due to humoral rejection (HR-TMA) or due to calcineurin inhibitor toxicity (CNI-TMA). Although the triggers are known, our knowledge about the thrombogenic transcriptome changes in the microvessels is rudimentary. Methods We examined the expression of several prothrombotic and antithrombotic genes in 25 biopsies with rTx-TMA (6 RecTMA, 9 HR-TMA, and 10 CNI-TMA) and 8 controls. RNA from microdissected glomeruli of paraffin-embedded tissue was isolated and mRNA transcripts were quantified with real-time polymerase chain reaction after preamplification. Results were correlated with clinicopathologic parameters. Results Glomerular mRNA expression of KLF2, KLF4, and tPA was lower and that of PAI-1 was higher in rTx-TMA than in the controls. Glomerular mRNA expression of KLF2 and KLF4 correlated with that of tPA and inversely with that of PAI-1 in rTx-TMA. The mRNA expression of complement regulators CD46 and CD59 were higher in rTx-TMA than in the controls. Only in HR-TMA were glomerular ADAMTS13 and CD55 down-regulated. Conclusions The glomerular capillary bed seems to contribute to all subtypes of rTx-TMA by down-regulation of the endothelial transcription factors KLF2 and KLF4, indicating dedifferentiation with subsequent up-regulation of PAI-1 and down-regulation of tPA, resulting in inhibition of local fibrinolysis. Decreased glomerular expression of ADAMTS13 and CD55 could be an additional pathway toward microthrombosis exclusively in HR-TMA.

Comprehensive analysis of glomerular mRNA expression of pro- and antithrombotic genes in atypical haemolytic-uremic syndrome (aHUS)

Atypical haemolytic–uremic syndrome (aHUS) is, in most cases, due to hereditary or acquired defects in complement regulation and a life-threatening disease. Despite the rapidly grown knowledge about the primary defects in aHUS, the pathogenesis that links complement dysregulation with microthrombus formation in aHUS is still unknown. Thus, we examined the glomerular microvascular expression of pro- and antithrombotic genes. Glomeruli were microdissected from 12 archival paraffin-embedded biopsies with aHUS and from seven control biopsies. Glomerular mRNA expression was quantified by single real-time PCR reactions after preamplification. In addition immunostains were performed for plasminogen activator inhibitor 1 (PAI-1) and for tissue plasminogen activator (tPA). Results were compared between cases and controls and with clinical data. Glomeruli in aHUS had increased mRNA expression of antifibrinolytic, prothrombotic PAI-1, antithrombotic thrombomodulin (THBD) and CD73 and decreased expression of profibrinolytic, antithrombotic tPA compared to controls. Impaired fibrinolysis due to increased microvascular expression of the antifibrinolytic PAI-1 in combination with the decreased expression of the profibrinolytic tPA seems to be a final common pathway in renal thrombotic microangiopathy that is also effective in aHUS. The concomitant induction of antithrombotic transcripts likely indicates counterregulatory efforts, demonstrating that the capillary bed is not a passive victim of complement attack. Future research should investigate if and how complement activation could induce the reported shift in the expression of PAI-1 and tPA.

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.

Arteriolar vascular smooth muscle cell differentiation in benign nephrosclerosis

BACKGROUND Benign nephrosclerosis (bN) is the most prevalent form of hypertensive damage in kidney biopsies. It is defined by early hyalinosis and later fibrosis of renal arterioles. Despite its high prevalence, very little is known about the contribution of arteriolar vascular smooth muscle cells (VSMCs) to bN. We examined classical and novel candidate markers of the normal contractile and the pro-fibrotic secretory phenotype of VSMCs in arterioles in bN. METHODS Sixty-three renal tissue specimens with bN and eight control specimens were examined by immunohistochemistry for the contractile markers caldesmon, alpha-smooth muscle actin (alpha-SMA), JunB, smoothelin and the secretory marker S100A4 and by double stains for caldesmon or smoothelin with S100A4. RESULTS Smoothelin immunostaining showed an inverse correlation with hyalinosis and fibrosis scores, while S100A4 correlated with fibrosis scores only. Neither caldesmon, alpha-SMA nor JunB correlated with hyalinosis or fibrosis scores. Cells in the arteriolar wall were exclusively positive either for caldesmon/smoothelin or S100A4. CONCLUSIONS This is the first systematic analysis of VSMC differentiation in bN. The results suggest that smoothelin is the most sensitive marker for the contractile phenotype and that S100A4 could be a novel marker for the secretory phenotype in vivo. The other markers did not seem to differentiate these phenotypes in bN. Thus, VSMC phenotype markers should be defined in the context of the vessel segment and disease under examination. S100A4 could not only be a marker of pro-fibrotic secretory VSMCs in bN but also an important mediator of arteriolar fibrosis.

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.

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.

Histology and mRNA Expression: Insights Into the Pathophysiology of Thrombus Formation and Glomerular Remodeling in Thrombotic Microangiopathy in Renal Transplants: 1927

Introduction: Metabolomics is a system approach used also to investigate the metabolic profile by multivariate data analysis tools. NMR spectroscopy is a analytical platform for biological matrices, it enables the quick generation of spectral profiles of low molecular weights metabolites. The aim of the work is to control the kidney graft recovery process in an non-invasive way, estimating the process by 1H-NMR spectroscopy and chemometrics on urine samples as they contains metabolites concerning the pathological and clinical state of the patients. Methods: In the study 15 patients (9 males and 6 females) have been followed during the kidney graft recovery process and the first follow up days. Samples have been collected every day, for each patient. In order to develop a model that was able to describe the recovery process from a general point of view, all the data from the patient cohort have been studied together. Results: The chemometric model is able to describe the whole cohort of patients, as each patients follow its own path to recovery, moving to one state to the other only when “time has come“. The model has as many clusters as the clinical states are, and it doesn‘t matter for how long each single patient stay into those clusters because we used a daily based sampling. In order to analyse the changes in metabolic pattern that are connected to the recovery process, we used the typical two steps analytical procedure (often used in metabolomics): first we use an unsupervised technique, Principal Components Analysis, to find trajectories and clustering, then we model the system by using a classification technique, Partial Least Square Discriminant Analysis. The global model, that reveal a clear three stage progress recovery, common to all the patients (figure 1), has been used as a reference model to asses anomalous patients behaviour. The model is made of a first stage, when the kidney is not still working, then a stage characterised by a recovery of the kidney capabilities and functions. These two stages are rather often clearly detached. The third stage is the follow up stage, in that case samples result to be far from the ones collected during the hospital stay. All those stages are characterised by well defined metabolic patterns.