14:00 - 15:30
Poster DGfN 2020
Experimentelle Nephrologie 6 (P195 - P202; LA42 - LA44)
Objective: The Wilms tumor transcription factor WT1 is necessary for kidney development. Identification of WT1 downstream target genes may thus allow one to analyze genetic pathways during nephrogenesis. Since mice with germline knockout of Wt1 are embryonic lethal and lack kidneys, alternative tools are needed to study WT1-dependent transcriptional networks. By combining vivo-morpholino induced gene knockdown in metanephric explants with next generation sequencing, we aimed at establishing a novel instrument to identify WT1 downstream targets in kidney development.
Method: Kidneys were excised from mouse embryos (E12.5) and grown ex vivo. The explants were treated for 48 h with either Wt1 antisense or mismatch vivo-morpholino. WT1 knockdown was assessed by immunoblotting, and pooled mRNAs were subjected to deep sequencing. The DESeq package was used to identify the differentially expressed genes. Deep sequencing data were validated by quantitative PCR of more than a dozen of arbitrarily picked transcripts.
Results: Silencing of Wt1 by vivo-morpholino treatment significantly reduced 308 and increased 258 mRNAs in metanephric organ cultures. Comparison with previously published ChIP-sequencing data (Motamedi et al., 2014) showed that WT1 binds to 217 of the down-regulated and 194 of the up-regulated genes in murine embryonic kidneys. Among these differentially expressed genes, 184 could be retrieved from the atlas of gene expression in the developing kidney (Brunskill et al, 2008). According to this atlas, 32 of the 184 genes are predominantly expressed in the metanephric mesenchyme, i.e. the site of WT1 expression. The endogenous mRNA levels of these top candidates were measured by quantitative PCR in cells treated with Wt1 siRNA or non-targeting siRNA. We used murine mesonephros-derived M15 cells, human U2OS osteosarcoma cells and renal clear cell carcinoma-derived 786-0 cells to analyze gene expression patterns. Subsequent co-transfection experiments using various reporter constructs indicated that the promoters of the homeobox b9 (Hoxb9), natriuretic peptide receptor 3 (Npr3) and dishevelled associated activator of morphogenesis 2 (Daam2), among other genes, are significantly stimulated by WT1.
Conclusion: With these findings, we identified sets of genes in murine embryonic kidneys that are regulated either directly or indirectly by WT1. In conclusion, combination of vivo-morpholino induced gene knockdown in embryonic kidney explants with deep RNA-sequencing is a useful approach for analyzing transcriptional networks in kidney development.
Objective: Hypoxia contributes to renal injury in acute and chronic kidney diseases. HIFs have an evident role in kidney injury and repair by activation of HIF target genes involved in the cellular adaptation to hypoxia. Accumulating data indicate that intrinsic HIFs activation can protect against renal damage. Recently we showed that reduced expression of the MAPK-organizer 1 (MORG1) in MORG1+/- mice improved the renal damage in animal models of T1- and T2-DM, and systemic hypoxia, partially by elevating HIFs expression through its complex with PHD3 protein. However, the mechanisms regulating MORG1 expression are not well understood. Here, we underwent studies to investigate the hMORG1 promoter and its regulation by hypoxia and PHDs pharmacological inhibitors in HEK293 cells.
Method: The hMORG1 promoter was amplified from HEK293 cells and sub-cloned into pGL2 basic vector. The influence of 1% O2 induced hypoxia and PHDs inhibitors 3,4-DHB and L-Mimosine on promoter activity was assayed by transient transfection of full length and truncated constructs of the hMORG1 promoter, followed by Luciferase assay.The impact of hypoxia and PHDs inhibitors on MORG1 promoter regulation was evaluated 24h post stimuli. The MORG1 expression was measured by real-time PCRs and Western blotting.
Results: Analyses of the hMORG1 promoter sequence revealed that there are 3 potential HRE binding sites. The wild-type promoter transcriptional activation was significantly elevated in HEK293 cells exposed to 500 µM 3,4-DHB or 500 µM L-Mimosine compared with controls. Removal of the two proximal HRE by truncation, reduced significantly the basal and the PHDs inhibitors dependent promoter activity. In agreement, both inhibitors elevated MORG1 mRNA and protein levels. In contrast, hypoxia inhibited the promoter transcriptional activity compared with normoxia. Low oxygen decreased significantly MORG1 mRNA and protein expression, while elevated Phd3 mRNA levels.
Conclusion: Accumulation of HIFs by PHDs inhibition in HEK293 cells induced the MORG1 transcriptional activity associated with increased MORG1 mRNA and protein levels. Interestingly, hypoxia demonstrated an opposing effect to the PHDs inhibitors on MORG1 promoter activity depicted by reduced MORG1 reporter activity and MORG1 mRNA and protein levels. These findings suggest that hypoxia and pharmacological stabilization of HIFs by PHDs inhibition regulate the MORG1 promoter activity by different mechanisms, which need to be further elucidated. The role of HIF-1a and HIF-2a TF on MORG1 promoter regulation will be studied by CHIP assays.
Hintergrund: Die Kalzifizierung und damit einhergehende Versteifung der Gefäßwand ist ein kardiovaskulärer Risikofaktor, insbesondere bei Patienten mit chronischer Niereninsuffizienz. Nach Nierentransplantation ist die kardiovaskuläre Mortalität der Patienten im Vergleich zu nierengesunden Menschen weiterhin erhöht, was teilweise auf den Einfluss von Immunsuppressiva zurückgeführt werden kann. Das Projektziel ist es, den Einfluss von Azathioprin (AZA) auf aortale glatte Gefäßmuskelzellen (VSMC) in einem Rattenmodell zu untersuchen.
Methode: In vitro Untersuchungen werden aortale VSMC der Ratte verwendet. Die in vivo Therapie mit AZA erfolgt über 24 Wochen in Ratten. Die mRNA Genexpression wird mittels quantitativer Real-time PCR bestimmt. Die Interleukin (IL)-1ß und IL-6 Plasmakonzentration wird mittels Bioplex bestimmt. Die 8-Oxo-2-desoxyguanosine (8-Oxo) Plasmakonzentration wird mittels ELISA gemessen.
Ergebnisse: Die Stimulation von VSMC mit 6-Mercaptopurin, dem aktiven Metaboliten von AZA, induziert in vitro die Mineralisierung und Seneszenz der Zellen. In vivo induziert AZA eine Zunahme des Kalziumgehaltes und damit der Kalzifizierung in der Aorta, nachgewiesen über die Quantifizierung des Gewebekalziumgehaltes und histologische Färbung mittels van Kossa. Des Weiteren werden typische Marker des Seneszenz-assoziierten Phänotyps (SASP) im aortalen Gewebe durch AZA signifikant stimuliert. Die mRNA Expression von cbfa1, einem osteogenen Transkriptionsfaktor, sowie seine regulierten Gene Alkalische Phosphatase (ALP) und Osteopontin (OPN) steigen signifikant unter AZA Therapie an, wohingegen die mRNA Expression von SM22a abnimmt. Des Weiteren steigt die Expression von IL-1ß und IL-6 im aortalen Gewebe signifikant an im Vergleich zu den Kontrolltieren. Dieser Effekt auf die IL-1ß und IL-6 Konzentration ist auch systemisch in den mit AZA behandelten Tiere im Vergleich zur Kontrollgruppe zu sehen. Durch den Abbau von AZA über die Xanthinoxidase (XO) nimmt der oxidative Stress (ROS) in den Tieren zu, gemessen mittels 8-Oxo-Plasmakonzentrationen. Die mRNA Genexpression der XO im aortalen Gewebe nimmt unter AZA Therapie zu. Ein XO-ROS-abhängiger Signalweg konnte auch in vitro bestätigt werden.
Zusammenfassung: Die in vivo Daten im Tiermodell bestätigen die Induktion der Mineralisierung der Gefäßwand und den damit verbundenden Einfluss einer immunsuppressiven Therapie mit AZA auf die Veränderung der Gefäßstruktur.
Objective: Focal and segmental glomerulosclerosis is a frequent histological pattern seen in nephrotic syndrome with only limited therapeutic interventions. During FSGS, podocytes are injured, foot processes effaced, parietal epithelial cells (PECs) activated which then form cellular lesions leading to progressive scarring. One approach to finding new therapeutics is to use high-content screenings, typically in in vitro models that unfortunately do not replicate glomerular morphology and physiology sufficiently. Due to its size, zebrafish larvae are the simplest vertebrate model for high-throughput screenings. Until today, it has been unclear whether zebrafish can develop FSGS.
Method: In the Tg(nphs2:GAL4), Tg(UAS:Eco.nfsb-mCherry) strain, podocytes express nitroreductase. Upon administration of 80 µM metronidazole, podocytes were partially injured. Proteinuria was in vivo quantified with i.v. injected high-molecular-weight fluorescent dextran. Larvae were assessed using classic histology and electron microscopy. Immunofluorescence staining was carried out to investigate extracellular matrix (laminin), proliferation (pcna), and parietal and tubular cell origin (pax2a, atp1a1).
Results: After partial podocyte-depletion, larvae developed severe nephrotic syndrome as shown by hypoproteinemic whole-body edema (Fig. A) and proteinuria indicated by the leakage of 500 kDa dextran through the filtration barrier. The ultrastructural evaluation showed global foot process effacement of remaining podocytes (Fig. D). Podocin was decreased in immunofluorescence on tissue sections. Moreover, we found several mammalian FSGS features: Sub-podocyte pseudocysts (asterisk in Fig. D), microvillous transformation, de novo formation of tight junctions as well as parietovisceral adhesions with Bowman’s capsule. We found PECs activated and changed towards a cuboidal phenotype. Pcna expression showed PEC-proliferation and pax2a staining PEC-recruitment to the glomerular tuft (arrowheads Fig. B). Most importantly, we found significant extracellular matrix deposition on the glomerular tuft as demonstrated by Jone’s silver staining and laminin immunofluorescence (Fig. C).
Conclusion: Herein we show that upon podocyte-depletion, zebrafish larvae develop functional and morphological features of human FSGS such as nephrotic syndrome, foot process effacement, PEC-activation and deposition of extracellular matrix on the glomerular tuft. We conclude that this model resembles FSGS in important features and therefore propose its applicability for a high-throughput drug screening assay.
Objective: The Western diet is rich in salt, which poses various health risks e.g. cardiovascular diseases or multiple sclerosis. A high salt diet (HSD) leads to a sodium accumulation in the skin and stimulates the immune system through the nuclear factor of activated T cells (Nfat5)-signaling pathway. Also the kidney medulla can store sodium to build an osmotic gradient for water conservation. Here, we studied the effect of an HSD on immune defense against bacterial pyelonephritis, the most common kidney infection.
Method: We used a murine model to study urinary tract infections (UTI) in mice, mimics the situation of pyelonephritis in humans. Mice were given normal-salt diet (NSD 0,3% Na) or high-salt diet (1,71% Na +0,9% NaCl autoclaved tap water) ad libitum for one week. Then, we instilled 1010 CFUs of uropathic E.coli into the bladder of anestetized female mice. The procedure was repeated 3 hours later. After 18 hours, we collected the kidneys and went for further analysis. This included determination of colony forming units (CFU) and histological analysis of the infected kidneys. Immune cells were stained with antibodies and identified via Flow Cytometry. RNA-Sequence data and RT-PCR results were performed to show differentially regulated genes in neutrophils under different diet forms. Changes of the horomone status were detected using ELISA assays.
Results: We found that the murine kidney did not accumulate sodium under an HSD. The accumulated sodium must be excreted, therefore the kidney used urea instead to build the osmotic gradient. Urea suppressed the antibacterial functionality of neutrophils and thereby aggravated the renal infection. As a second consequence of enhanced sodium supply, we found that mineralocorticoid production was reduced via suppressed aldosterone synthase. This caused an accumulation of aldosterone precursors with glucocorticoid functionality, which abolished the diurnal adrenocorticotropic hormone (ACTH)-driven glucocorticoid rhythm. As a consequence, neutrophil development was compromised and their antibacterial functionality was impaired systemically.
Finally, healthy humans consuming an HSD for one week showed hyperglucocorticoidism and impaired anti-bactericidal neutrophil function as well.
Conclusion: In summary, an HSD suppresses intrarenal neutrophils by altering the local microenvironment, and systemically by glucocorticoid-mediated immunosuppression. These findings argue against high salt consumption during bacterial infections.
Objective: Developing, refining and deploying new drugs to target specific molecules in cell types and tissues of interest is crucial for advancing towards precision medicine. Antisense oligonucleotides (ASO) are chemically synthesized, short DNA oligomers that can be applied to target specific mRNAs via hybridization and degradation, thus resulting in a reduced synthesis of related proteins. Recent data suggest that newer, more stable and effective generations of ASO may be of therapeutic value, including in humans. However, our understanding of the biodistribution and efficacy of systemically delivered ASO in organs is insufficient.
Method: Here, we investigated the distribution, cell incorporation and gene knockdown efficiency of ASO specifically in kidney cells in vitro and in vivo using an unbiased approach.
Results: First, we designed and generated several eGFP-specific LNA Gapmer-type ASO and tested for eGFP knockdown potency in vitro. Promising candidates were amplified and then used in two transgenic mouse models of ubiquitous, constitutive eGFP-expression. EGFP-mice were i.p. injected with ASO directed against eGFP or control ASO for five to ten days, sacrificed and organs harvested for analyses. Comprehensive immunohistological studies in kidney using cell-specific staining techniques found distinct and robust eGFP suppression in proximal tubular epithelial cells (PTEC) but not in other renal cell types including the TAL, collecting duct, interstitium and endothelial cells. To study the biodistribution and dynamics of systemic ASO in more detail, we administered fluorophorelabeled ASO to wildtype mice and found overwhelming ASO enrichment in PTEC, suggesting highly preferential ASO incorporation in this segment of the nephron. In line, temporary exposure of mammal PTEC to labeled ASO in vitro resulted in rapid and sustained increases in intracellular fluorescence, indicating efficient ASO uptake by PTEC.
Conclusion: In summary, our data provide strong evidence for a differentiated biodistribution and cellular internalization of ASO in renal tissue with systemic delivery, implicating important differences in ASO-mediated knockdown efficacy and favoring, overall, PTEC as potential target cells. This disparity in ASO accessibility and hence expectable impact needs to be considered when defining therapeutic targets in kidney.
Objective: Genome-wide association studies of metabolite concentrations have identified common genetic variants influencing urine metabolite levels. Gene-based tests study the aggregated impact of rare variants, and may reveal further associations. We therefore used this complementary approach to find and characterize additional genes influencing metabolite levels in urine.
Method: We examined 4,864 participants of the GCKD study with 226,233 genetic variants genotyped on the Illumina Exome Chip and 1,487 urine metabolites quantified from non-targeted mass spectrometry (Metabolon) and 53,714 metabolite ratios. Two gene-based tests (burden and sequence kernel association test) were carried out for each gene and each metabolite. For each gene, the aggregated effect of variants with minor allele frequency <1% and likely to be functional (splicing, nonsynonymous, stopgain and stoploss) was assessed, adjusted for sex, age, eGFR and UACR. Bonferroni-corrected statistical significance was 1.5E-09 (metabolites) and 4.0E−11 (ratios). A link to inborn errors of metabolism (IEMs) was assessed through literature review and in silico constraint-based modeling of knockouts of significant genes in a virtual whole-body, organ-resolved metabolic human, followed by expression and enrichment analyses.
Results: We identified 53 metabolite-gene pairs involving 26 genes and 75 ratio-gene pairs involving 7 genes. Of 30 unique genes identified in total, 16 are known to underlie recessively inherited IEMs, with phenotypes of affected patients matching the associated metabolites or ratios identified in our study. The encoded genes were significantly enriched for high expression in liver and kidney across 54 tissues, and especially for shared expression in both (OR=65, FDR=3e-7). Single-cell RNA-sequencing from human kidney and liver revealed proximal tubule cells and hepatocytes as the driving cell types. Use of whole-exome sequencing data in the UK Biobank allowed for linking genes to diseases that could plausibly be explained by the identified metabolites. The in silico constraint-based modeling of knockouts of the implicated genes correctly predicted the direction of metabolite changes in urine and blood that were observed in gene-based tests.
Conclusion: Our study extends the map of genes influencing urine metabolite levels, illuminates ongoing metabolic processes at the systemic and organ level, implicates novel candidate variants and genes for IEMs, and highlights the potential of linking population genetics to whole-body, organ-resolved models of human metabolism.
Objective: Endothelial dysfunction (ED) contributes to the high incidence of cardiovascular (CV) events in renal patients. One aspect of ED is the breakdown and changes in nanomechanics of the endothelial glycocalyx (eGC). Clinical research reported that the phosphate hormone FGF23 is highly upregulated in kidney patients which associated with CV pathologies. Recently, several studies highlighted the different effects of c-terminal FGF23 (cFGF23) vs. intact FGF23 (iFGF23). The aim of this study was to find the underlying mechanisms of either cFGF23 or iFGF23 on eGC damage.
Method: Primary human umbilical vein endothelial cells (HUVEC) were cultured in medium until confluence. Subsequently, uremic sera from 12 renal patients with high (>30ng/mL) vs. low (<2ng/mL) cFGF23 levels were added to the medium at 10% vs. serum from healthy individuals. Using Atomic Force Microscopy (AFM), height and stiffness of the eGC were measured. In further experiments, HUVEC stimulation with iFGF23 or cFGF23 (each 30ng/mL) was performed for one hour. Next to AFM measurements, eGC quantification (WGA surface stainings) and ELISA of the supernatant was performed to detect the eGC substance syndecan-1. Unstimulated cells were taken as control. Mann-Whitney U test or unpaired t-test was used to compare groups.
Results: AFM measurements of uremic patient sera revealed a 14% decrease in eGC height and 8% stiffness increase with high cFGF23 levels compared to low levels (each P<0.0001). Upon stimulation with cFGF23, the height of the eGC was reduced (9%; P=0.0019) and stiffness upregulated compared to healthy samples (14%; P<0.0001). eGC quantification and syndecan-1 levels in the supernatants were unchanged. Stimulation with iFGF23 did not show an effect on the nanomechanical properties. Instead, the eGC amount was downregulated (11%; P<0.0001) and syndecan-1 levels were increased (12%; P=0.0347).
Conclusion: Our data suggest that FGF23 contributes to the development of ED. For cFGF23, all results suggest collapse of the eGC as indicated by decreased height, increased stiffness, less eGC amount and the absence of shed syndecan-1. In contrast, iFGF23 stimulation seemed to cause shedding of the eGC with unchanged height, less surface eGC and increased syndecan-1 levels. We conclude FGF23 to trigger eGC damage and thus, breakdown of the vasoprotective barrier contributing to CV events in renal patients.
Objective: Over recent decades, concerns regarding the potential risk of plastic particles on the environment and human health have been increased. As we know very little about the particle accumulation in the food chain, and also their further long-term effect for the human health, in this work, we evaluated the primary effects of four different types of plastic particles (polyvinyl chloride (PVC), polypropylene (PP), polyamide (PA) and tyre wear particles) of different sizes on the human podocytes as a representative kidney cells.
Method: To that aim, we applied different biological assays and imaging techniques including; bright field imaging, cell viability test and phalloidin staining. Additionally, the particles uptake by the cells and the further changes in their biological features were visualized with the use of Scanning Electron Microscopy (SEM).
Results: As a primary results, the cytotoxicity response of particle treatment is found to be dependend on the polymer type. As an example higher concentration of PP particle as compared to PVC, PA, and tyre wear cause a similar rate of cell mortality. Furthermore, the degree of particle attachment on the surface of the cells depends on their adhesion properties. Some particles like PVC, PA and Tyre wear particles remained attached to the cell surface even after two-three times of washing with PBS.
Conclusion: This study suggests that exposure duration and particle concentrations are two of the key factors to evaluate the toxicological effect of particle on podocytes as a highly-specialized epithelial cells in the kidney.It is supposed that, two mechanisms can be related to the plastic particles effects on podocytes and their final death. First, their attachment on the cell surface and following that the limitation of nutrient uptake by the cells. Second, uptake of smaller size of particles into the cells through the phagocytosis process.
Objective: Adverse events during pregnancy leading to a reduction in birth weight due to intrauterine growth restriction (IUGR) increase the risk for hypertension and cardiovascular disease, stroke, diabetes mellitus, obstructive pulmonary disease or chronic kidney disease (CKD) later in life (fetal origin of adult disease theory or Barker’s hypothesis). The kidneys of such low birth weight (LBW) infants are characterized by a reduced number of nephrons. However the exact mechanisms whereby reduced nephron endowment drives the onset and progression of CKD remain to be defined. IUGR is associated with numerous unfavourable gestational conditions, including sociodemographic, genetic and environmental factors, i.e. intrauterine hypoxia. The latter can stem from multiple aetiologies including umbilical cord obstruction, smoking, but also high altitude, which affects more than 2% of the world’s population. Hypoxia activates a tightly controlled signalling cascade by stabilisation of the hypoxia-inducible transcription factor (HIF) alpha and transactivation of target genes.
Method: In this study, we set up and validate a robust mouse model of fetal hypoxia-induced IUGR by exposing gravid mice to normobaric hypoxia (mimicking high elevation), and characterize its short and long-term consequences, using the kidney as a readout.
Results: We show that hypoxia induces an ectopic expression profile of liver-specific genes in fetal kidneys. Of these, we identified the plasma glycoprotein fetuin-A (Ahsg) as a novel, evolutionary conserved HIF target gene, and further investigated its role in hypoxic fetal kidneys using a KO approach. Beyond its recognized role as systemic calcification inhibitor, our findings establish fetuin-A as a local calcium mineral scavenger, not only counteracting intrarenal calcification, but also attenuating renal inflammation, macrophage activation and fibrotic remodelling. Furthermore, fetuin-A deficiency during fetal hypoxia leads to enhanced proteinuria and reduced glomerular filtration in adult animals.
Conclusion: These results provide solid mechanistic proof of Barker’s hypothesis, highlighting the role of the novel HIF target gene fetuin-A to maintain tissue integrity during development and its protective effect against the progression of renal fibrosis. Our study thus paves the way to novel therapeutic approaches mitigating mineral stress-induced inflammation and damage in soft tissues.
Objective: The Protein tyrosine phosphatase receptor type O (PTPRO) is present on the apical cell surface of podocytes. It is associated with the WNT/-catenin pathway and the NF-B pathway. Mutations in PTPRO cause childhood onset steroid resistant nephrotic syndrome. Absence of PTPRO was observed in primary FSGS and the podocyte structure is altered in PTPRO knock out mice. We recently detected an upregulation of microRNA-26b-5p (miR-26b) in urine samples from patients with FSGS. PTPRO is a potential target of miR-26b. Here, we investigated the regulation of podocyte PTPRO and its dependence on miR-26b.
Method: Expression and regulation of PTPRO was investigated in immortalized cultured human podocytes after treatment with TGF-, Dexamethasone and Puromycin, as well as after transfection with a miR-26b mimic or a PTPRO siRNA, by qPCR, Western Blot and Immunofluorescence.
Results: PTPRO was upregulated after stimulation with TGF- and Dexamethasone. In contrast, transfection with a miR-26b mimic and treatment with Puromycin, which is a model for FSGS, decreased PTPRO expression. Immunofluorescence staining showed a stress inducted alteration of podocyte actin cytoskeleton after PTPRO knockdown. Dexamethasone was able to rescue Puromycin induced PTPRO knockdown.
Conclusion: We could show a down regulation of PTPRO after Puromycin treatment that serves as a model for FSGS as well as after transfection with miR-26b that is overexpressed in FSGS patients. Dexamethasone treatment might be a therapeutic option especially for FSGS patients with reduced PTPRO.
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