Alteration of podocyte phenotype in the urine of women with preeclampsia.
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ABSTRACT:
SUBMITTER: Zhai T
PROVIDER: S-EPMC4823657 | biostudies-other | 2016 Apr
REPOSITORIES: biostudies-other
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Project description:Background and objectivesPreeclampsia is characterized by hypertension and proteinuria, and increased shedding of podocytes into the urine is a common finding. This finding raises the question of whether preeclamptic nephropathy involves podocyte damage. This study examined podocyte-related changes in a unique sample of renal tissues obtained from women who died of preeclampsia.Design, setting, participants, & measurementsAll patients with preeclampsia who died in The Netherlands since 1990 and had available autopsy tissue were identified using a nationwide database of the Dutch Pathology Registry (PALGA). This resulted in a cohort of 11 women who died from preeclampsia. Three control groups were also identified during the same time period, and consisted of normotensive women who died during pregnancy (n=25), and nonpregnant controls either with (n=14) or without (n=13) chronic hypertension. Glomerular lesions, including podocyte numbers, podocyte proliferation, and parietal cell activation, were measured.ResultsPatients with preeclampsia had prominent characteristic glomerular lesions. The results showed that the number of podocytes per glomerulus did not differ significantly between the patients with preeclampsia and the control groups. However, preeclampsia was associated with a significant increase in intraglomerular cell proliferation (7.3% [SD 9.4] of the glomeruli of patients with preeclampsia had Ki-67-positive cells versus 1.6% [SD 3.3] of the glomeruli of hypertensive controls and 1.1% [SD 1.3] of nonpregnant controls; P=0.004) and activated parietal epithelial cells on a podocyte location (34% [SD 13.1] of the glomeruli of patients with preeclampsia versus 18.0% [SD 15.3] of pregnant controls, 11.9% [SD 13.2] of hypertensive controls, and 10.8% [SD 13.4] of nonpregnant controls; P=0.01).ConclusionsThese findings suggest that the recently described mechanisms of podocyte replacement play a role in preeclampsia. These results provide key new insights into the pathogenesis of preeclamptic nephropathy, and they open new possibilities for developing therapeutic modalities.
| S-EPMC4123409 | biostudies-literature
Project description:BackgroundLevels of urinary microvesicles, which are increased during various kidney injuries, have diagnostic potential for renal diseases. However, the significance of urinary microvesicles as a renal disease indicator is dampened by the difficulty to ascertain their cell source.ObjectivesThe aim of this study was to demonstrate that podocytes can release migrasomes, a unique class of microvesicle with size ranging between 400 and 2,000 nm, and the urine level of migrasomes may serve as novel non-invasive biomarker for early podocyte injury.MethodIn this study, immunofluorescence labeling, electronic microscopy, nanosite, and sequential centrifugation were used to purify and analyze migrasomes.ResultsMigrasomes released by podocytes differ from exosomes as they have different content and mechanism of release. Compared to podocytes, renal tubular cells secrete markedly less migrasomes. Moreover, secretion of migrasomes by human or murine podocytes was strongly augmented during podocyte injuries induced by LPS, puromycin amino nucleoside (PAN), or a high concentration of glucose (HG). LPS, PAN, or HG-induced podocyte migrasome release, however, was blocked by Rac-1 inhibitor. Strikingly, a higher level of podocyte migrasomes in urine was detected in mice with PAN-nephropathy than in control mice. In fact, increased urinary migrasome number was detected earlier than elevated proteinuria during PAN-nephropathy, suggesting that urinary migrasomes are a more sensitive podocyte injury indicator than proteinuria. Increased urinary migrasome number was also detected in diabetic nephropathy patients with proteinuria level <5.5 g/day.ConclusionsOur findings reveal that podocytes release the "injury-related" migrasomes during migration and provide urinary podocyte migrasome as a potential diagnostic marker for early podocyte injury.
| S-EPMC7706499 | biostudies-literature
Project description:Apolipoprotein L1 (APOL1)-miR193a axis has been reported to play a role in the maintenance of podocyte homeostasis. In the present study, we analyzed transcription factors relevant to miR193a in human podocytes and their effects on podocytes' molecular phenotype. The motif scan of the miR193a gene provided information about transcription factors, including YY1, WT1, Sox2, and VDR-RXR heterodimer, which could potentially bind to the miR193a promoter region to regulate miR193a expression. All structure models of these transcription factors and the tertiary structures of the miR193a promoter region were generated and refined using computational tools. The DNA-protein complexes of the miR193a promoter region and transcription factors were created using a docking approach. To determine the modulatory role of miR193a on APOL1 mRNA, the structural components of APOL1 3' UTR and miR193a-5p were studied. Molecular Dynamic (MD) simulations validated interactions between miR193a and YY1/WT1/Sox2/VDR/APOL1 3' UTR region. Undifferentiated podocytes (UPDs) displayed enhanced miR193a, YY1, and Sox2 but attenuated WT1, VDR, and APOL1 expressions, whereas differentiated podocytes (DPDs) exhibited attenuated miR193a, YY1, and Sox2 but increased WT1, VDR, APOL1 expressions. Inhibition of miR193a in UPDs enhanced the expression of APOL1 as well as of podocyte molecular markers; on the other hand, DPD-transfected with miR193a plasmid showed downing of APOL1 as well as podocyte molecular markers suggesting a causal relationship between miR193a and podocyte molecular markers. Silencing of YY1 and Sox2 in UPDs decreased the expression of miR193a but increased the expression of VDR, and CD2AP (a marker of DPDs); in contrast, silencing of WT1 and VDR in DPDs enhanced the expression of miR193a, YY1, and Sox2. Since miR193a-downing by Vitamin D receptor (VDR) agonist not only enhanced the mRNA expression of APOL1 but also of podocyte differentiating markers, suggest that down-regulation of miR193a could be used to enhance the expression of podocyte differentiating markers as a therapeutic strategy.
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Project description:miRNA microarray was performed for migrasomes and exosomes derived from urine
2020-05-14 | GSE150459 | GEO
Project description:Evidence suggests that loss of podocytes into urine contributes to development of glomerular diseases; shed podocytes are frequently viable and proliferate in culture conditions. To determine the phenotypic characteristics of viable urinary cells derived from human subjects, we established long-term urinary cell culture from two patients with focal segmental glomerulosclerosis and two healthy volunteers, via transformation with the thermosensitive SV40 large T antigen (U19tsA58) together with human telomerase (hTERT). Characterization of arbitrarily selected two clonal cell lines from each human subject was carried out. mRNA expression for the podocyte markers synaptopodin, nestin, and CD2AP were detected in all eight clones. Podocin mRNA was absent from all eight clones. The expression of nephrin, Wilms' tumor 1 (WT1), and podocalyxin mRNA varied among the clones, which may be due to transformation and/or cloning. These results suggest that podocyte cell lines can be established consistently from human urine. The generation of podocyte cell lines from urine of patients and healthy volunteers is novel and will help to advance studies of podocyte cell biology. Further improvements in the approaches to cell transformation and/or cell culture techniques are needed to allow cultured podocytes to fully reproduce in vivo characteristics.
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Project description:Renal histologic expression of the podocyte-specific protein, nephrin, but not podocin, is reduced in preeclamptic compared with normotensive pregnancies. We hypothesized that renal expression of podocyte-specific proteins would be reflected in urinary extracellular vesicles (EVs) of podocyte origin and accompanied by increased urinary soluble nephrin levels (nephrinuria) in preeclampsia. We further postulated that podocyte injury and attendant formation of EVs are related mechanistically to cellfree fetal hemoglobin (HbF) in maternal plasma. Our study population included preeclamptic (n=49) and normotensive (n=42) pregnant women recruited at delivery. Plasma measurements included HbF concentrations and concentrations of the endogenous chelators haptoglobin, hemopexin, and α1- microglobulin. We assessed concentrations of urinary EVs containing immunologically detectable podocyte-specific proteins by digital flow cytometry and measured nephrinuria by ELISA. The mechanistic role of HbF in podocyte injury was studied in pregnant rabbits. Compared with urine from women with normotensive pregnancies, urine from women with preeclamptic pregnancies contained a high ratio of podocin-positive to nephrin-positive urinary EVs (podocin+ EVs-to-nephrin+ EVs ratio) and increased nephrinuria, both of which correlated with proteinuria. Plasma levels of hemopexin, which were decreased in women with preeclampsia, negatively correlated with proteinuria, urinary podocin+ EVs-to-nephrin+ EVs ratio, and nephrinuria. Administration of HbF to pregnant rabbits increased the number of urinary EVs of podocyte origin. These findings provide evidence that urinary EVs are reflective of preeclampsia-related altered podocyte protein expression. Furthermore, renal injury in preeclampsia associated with an elevated urinary podocin+ EVs-to-nephrin+ EVs ratio and may be mediated by prolonged exposure to cellfree HbF.
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Project description:Differential proteomic technology was used to identify urine proteomic profile of gestational hypertension and preeclampsia. Urine samples were collected from 10 patients with gestational hypertension, 10 patients with mild preeclampsia, 10 patients with severe preeclampsia and 10 normal pregnancies and analyzed by 2‑D difference gel electrophoresis, then matrix assisted laser desorption ionization mass spectrometry was used to identify differential proteins. Subsequently, ELISA was used to verify the content variation of the identified proteins in 200 urine samples. In total, 30 differential proteins were identified. For prostaglandin‑H2 D‑isomerase (L‑PGDS), perlecan and other 15 proteins, the contents in patients with gestational hypertension were higher than that of normal pregnancies, but lower in mild and severe preeclampsia. By contrast, serum albumin and α‑1‑antitrypsin was lower in samples from patients with gestational hypertension and higher in patients with mild and severe preeclampsia compared with normal pregnancies. ELISA verified that the urinary concentration of L‑PGDS and perlecan were significantly lower in patients with preeclampsia than in normal pregnancies (P<0.05). Urine proteomics is a useful tool to identify potential biomarkers to distinguish between different types of hypertensive disorders in pregnancy. L‑PGDS and perlecan could potentially be used as markers to reflect the state of renal function, and may participate in the genesis and development of renal injury during preeclampsia.
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Project description:Mitotic catastrophe (MC) is a major cause of podocyte loss in vitro and in vivo. We evaluated urine samples (n = 184 urine samples from diabetic patients; n = 41 patients) from diabetic patients and determined the presence of podocytes in the urine and studied their characteristics, specifically asking whether apoptosis versus MC is present. We also evaluated diabetic glomeruli in renal biopsy specimens by electron microscopy (n = 54). A battery of stains including the antibody to podocalyxin (PCX) were used. PCX and podocytes (PCX+podo) showed nuclear morphologies such as a i) mononucleated normal shape (8.7%), ii) large and abnormal shape (3.8%), iii) multinucleated with or without micronucleoli (31.2%), iv) mitotic spindles (8.2%), v) single nucleus and denucleation combined (10.3%), and vi) denucleation only (37.0%). Large size/abnormal shape, multinucleation, mitotic spindles, and a combination of single nucleus and denucleation were considered features of MC (53.5%). Dual staining of PCX+podo was positive for Glepp 1 (50%), whereas none of PCX+podo were positive for nephrin, podocin, leukocyte, or parietal epithelial cell markers (cytokeratin 8), annexin V, cleaved caspase-3, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Ten percent of PCX+podo were positive for phosphorylated vimentin. Electron microscopy identified cellular and nuclear podocyte changes characteristic of MC. The majority of urine podocytes in diabetic patients showed MC, not apoptosis. This noninvasive approach may be clinically useful in determining progressive diabetic nephropathy or response to therapeutic intervention.
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Project description:Vitamin D deficiency is common in pregnant women and may contribute to adverse events in pregnancy such as preeclampsia (PET). To date, studies of vitamin D and PET have focused primarily on serum concentrations vitamin D, 25-hydroxyvitamin D3 (25(OH)D3) later in pregnancy. The aim here was to determine whether a more comprehensive analysis of vitamin D metabolites earlier in pregnancy could provide predictors of PET. Using samples from the SCOPE pregnancy cohort, multiple vitamin D metabolites were quantified by liquid chromatography-tandem mass spectrometry in paired serum and urine prior to the onset of PET symptoms. Samples from 50 women at pregnancy week 15 were analysed, with 25 (50%) developing PET by the end of the pregnancy and 25 continuing with uncomplicated pregnancy. Paired serum and urine from non-pregnant women (n?=?9) of reproductive age were also used as a control. Serum concentrations of 25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 24,25(OH)2D3 and 3-epi-25(OH)D3 were measured and showed no significant difference between women with uncomplicated pregnancies and those developing PET. As previously reported, serum 1,25(OH)2D3 was higher in all pregnant women (in the second trimester), but serum 25(OH)D2 was also higher compared to non-pregnant women. In urine, 25(OH)D3 and 24,25(OH)2D3 were quantifiable, with both metabolites demonstrating significantly lower (P?<?0.05) concentrations of both of these metabolites in those destined to develop PET. These data indicate that analysis of urinary metabolites provides an additional insight into vitamin D and the kidney, with lower urinary 25(OH)D3 and 24,25(OH)2D3 excretion being an early indicator of a predisposition towards developing PET.
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Project description:Preeclampsia is a complex genetic disorder with an incompletely understood pathogenesis. Its phenotype may be better elucidated by integrating symptoms. This study aimed to identify symptoms by gestational age and associations with novel preeclampsia candidate genes. Women with a history of preeclampsia recruited from The Preeclampsia Registry completed clinical/demographic, symptom surveys and provided medical records. DNA extracted from saliva was processed with multiplexed assays for eight single-nucleotide polymorphisms (SNPs) selected to tag candidate genes and/or located in symptom susceptibility regions. Groups with versus without symptoms were compared using χ2. Associations between SNPs and symptoms were analyzed as genotype categories and presence/absence of the variant allele. Logistic regression modeling was conducted with exploratory p = .05. In 114 participants, 113 reported at least 1 of the 18 symptoms. Symptoms varied by trimester. Nine symptoms were associated with seven SNPs. Visual disturbances were associated with three SNPs and nausea/vomiting with two SNPs. Modeling adjustment for maternal age and parity resulted in 15 associations between 9 symptoms and 8 SNPs. Medical records demonstrated 100% concordance with self-reported diagnosis and 48% concordance with reported severity. Findings indicated novel symptom-genotype associations in preeclampsia. The small sample was self-selected, but results support future studies including medical records review. When validated, these results may lead to holistic phenotyping of women to characterize subsets of preeclampsia. This approach may optimize health in pregnancy and later life for mothers and offspring through prediction, prevention, and precision nursing care.
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