RESUMEN
TGF-ß is considered an important cytokine in the development of interstitial fibrosis in chronic kidney disease. The TGF-ß co-receptor endoglin (ENG) tends to be upregulated in kidney fibrosis. ENG has two membrane bound isoforms generated via alternative splicing. Long-ENG was shown to enhance the extent of renal fibrosis in an unilateral ureteral obstruction mouse model, while short-ENG inhibited renal fibrosis. Here we aimed to achieve terminal intron retention of endoglin using antisense-oligo nucleotides (ASOs), thereby shifting the ratio towards short-ENG to inhibit the TGF-ß1-mediated pro-fibrotic response. We isolated mRNA from kidney biopsies of patients with chronic allograft disease (CAD) (n = 12) and measured total ENG and short-ENG mRNA levels. ENG mRNA was upregulated 2.3 fold (p < 0.05) in kidneys of CAD patients compared to controls, while the percentage short-ENG of the total ENG mRNA was significantly lower (1.8 fold; p < 0.05). Transfection of ASOs that target splicing regulatory sites of ENG into TK173 fibroblasts led to higher levels of short-ENG (2 fold; p < 0.05). In addition, we stimulated these cells with TGF-ß1 and measured a decrease in upregulation of ACTA2, COL1A1 and FN1 mRNA levels, and protein expression of αSMA, collagen type I, and fibronectin. These results show a potential for ENG ASOs as a therapy to reduce interstitial fibrosis in CKD.
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Endoglina , Fibrosis , Intrones , Riñón , Oligonucleótidos Antisentido , Factor de Crecimiento Transformador beta1 , Humanos , Endoglina/metabolismo , Endoglina/genética , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/genética , Intrones/genética , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Riñón/metabolismo , Riñón/patología , Masculino , Fibronectinas/metabolismo , Fibronectinas/genética , Femenino , Actinas/metabolismo , Actinas/genética , Persona de Mediana Edad , Animales , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Empalme Alternativo , Fibroblastos/metabolismo , Fibroblastos/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratones , Línea CelularRESUMEN
PURPOSE: To describe a recessively inherited cerebral small vessel disease, caused by loss-of-function variants in Nitrilase1 (NIT1). METHODS: We performed exome sequencing, brain magnetic resonance imaging, neuropathology, electron microscopy, western blotting, and transcriptomic and metabolic analyses in 7 NIT1-small vessel disease patients from 5 unrelated pedigrees. RESULTS: The first identified patients were 3 siblings, compound heterozygous for the NIT1 c.727C>T; (p.Arg243Trp) variant and the NIT1 c.198_199del; p.(Ala68∗) variant. The 4 additional patients were single cases from 4 unrelated pedigrees and were all homozygous for the NIT1 c.727C>T; p.(Arg243Trp) variant. Patients presented in mid-adulthood with movement disorders. All patients had striking abnormalities on brain magnetic resonance imaging, with numerous and massively dilated basal ganglia perivascular spaces. Three patients had non-lobar intracerebral hemorrhage between age 45 and 60, which was fatal in 2 cases. Western blotting on patient fibroblasts showed absence of NIT1 protein, and metabolic analysis in urine confirmed loss of NIT1 enzymatic function. Brain autopsy revealed large electron-dense deposits in the vessel walls of small and medium sized cerebral arteries. CONCLUSION: NIT1-small vessel disease is a novel, autosomal recessively inherited cerebral small vessel disease characterized by a triad of movement disorders, massively dilated basal ganglia perivascular spaces, and intracerebral hemorrhage.
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Hemorragia Cerebral , Enfermedades de los Pequeños Vasos Cerebrales , Trastornos del Movimiento , Linaje , Humanos , Femenino , Masculino , Enfermedades de los Pequeños Vasos Cerebrales/genética , Enfermedades de los Pequeños Vasos Cerebrales/patología , Enfermedades de los Pequeños Vasos Cerebrales/diagnóstico por imagen , Persona de Mediana Edad , Hemorragia Cerebral/genética , Hemorragia Cerebral/patología , Hemorragia Cerebral/diagnóstico por imagen , Trastornos del Movimiento/genética , Trastornos del Movimiento/patología , Trastornos del Movimiento/diagnóstico por imagen , Imagen por Resonancia Magnética , Alelos , Adulto , Anciano , Sistema Glinfático/patología , Sistema Glinfático/diagnóstico por imagen , Secuenciación del Exoma , Encéfalo/patología , Encéfalo/diagnóstico por imagen , Aminohidrolasas/genéticaRESUMEN
Initial cysts that are formed upon Pkd1 loss in mice impose persistent stress on surrounding tissue and trigger a cystic snowball effect, in which local aberrant PKD-related signaling increases the likelihood of new cyst formation, ultimately leading to accelerated disease progression. Although many pathways have been associated with PKD progression, the knowledge of early changes near initial cysts is limited. To perform an unbiased analysis of transcriptomic alterations in the cyst microenvironment, microdomains were collected from kidney sections of iKsp-Pkd1del mice with scattered Pkd1-deletion using Laser Capture Microdissection. These microdomains were defined as F4/80-low cystic, representing early alterations in the cyst microenvironment, F4/80-high cystic, with more advanced alterations, or non-cystic. RNA sequencing and differential gene expression analysis revealed 953 and 8088 dysregulated genes in the F4/80-low and F4/80-high cyst microenvironment, respectively, when compared to non-cystic microdomains. In the early cyst microenvironment, several injury-repair, growth, and tissue remodeling-related pathways were activated, accompanied by mild metabolic changes. In the more advanced F4/80-high microdomains, these pathways were potentiated and the metabolism was highly dysregulated. Upstream regulator analysis revealed a series of paracrine factors with increased activity in the early cyst microenvironment, including TNFSF12 and OSM. In line with the upstream regulator analysis, TWEAK and Oncostatin-M promoted cell proliferation and inflammatory gene expression in renal epithelial cells and fibroblasts in vitro. Collectively, our data provide an overview of molecular alterations that specifically occur in the cyst microenvironment and identify paracrine factors that may mediate early and advanced alterations in the cyst microenvironment.
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Quistes , Enfermedades Renales Poliquísticas , Ratones , Animales , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/metabolismo , Riñón/metabolismo , Perfilación de la Expresión Génica , Quistes/genética , Microambiente TumoralRESUMEN
Transmembrane protein 14A (TMEM14A) is a relatively unknown protein that is now identified to be required for maintaining the integrity of the glomerular filtration barrier. It is an integral transmembrane protein of 99 amino acids with three transmembrane domains. TMEM14A has been implied to suppress Bax-mediated apoptosis in other studies. Other than that, little is currently known of its function. Here, we show that its expression is diminished before onset of proteinuria in a spontaneously proteinuric rat model. Knocking down tmem14a mRNA translation results in proteinuria in zebrafish embryos without affecting tubular reabsorption. Also, it is primarily expressed by podocytes. Lastly, an increase in glomerular TMEM14A expression is exhibited in various proteinuric renal diseases. Overall, these results suggest that TMEM14A is a novel factor in the protective mechanisms of the nephron to maintain glomerular filtration barrier integrity.
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Proteínas Reguladoras de la Apoptosis , Barrera de Filtración Glomerular , Proteínas de la Membrana , Podocitos , Animales , Ratas , Glomérulos Renales/metabolismo , Podocitos/metabolismo , Proteinuria/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de la Membrana/genética , Proteínas Reguladoras de la Apoptosis/genéticaRESUMEN
Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns-/- zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS-/- kidney cells and injection into ctns-/- zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns-/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns-/- larvae, and restoration of the zebrafish pronephros function.
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Sistemas de Transporte de Aminoácidos Neutros , Cistinosis , Animales , Cistinosis/genética , Cistinosis/terapia , Cistina/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , ARN Mensajero/genética , ARN Mensajero/uso terapéutico , Modelos Teóricos , Suplementos Dietéticos , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismoRESUMEN
Metabolic reprogramming is a driver of autosomal dominant polycystic kidney disease (ADPKD) progression and a potential therapeutic intervention route. We showed before that the AMP-associated protein kinase (AMPK) activator salsalate attenuates cystic disease progression. Here, we aim to study the early, direct effects of short salsalate treatment in adult-onset conditional Pkd1 deletion mice. Cystic mice were treated with salsalate for two weeks, after which NMR metabolomics and RNA sequencing analyses were performed. Pkd1 deletion resulted in clear metabolomic dysregulation. Short salsalate treatment has small, but significant, effects, reverting acetylcarnitine and phosphocholine concentrations back to wildtype levels, and showing associations with altered purine metabolism. RNA sequencing revealed that short salsalate treatment, next to restoring energy metabolism toward wildtype levels, also affects cell proliferation and inflammation, in PKD. We show that salsalate positively affects major dysregulated processes in ADPKD: energy metabolism, cell proliferation, and inflammation, providing more insights into its working mechanisms.
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Multiple sclerosis (MS) pathology features autoimmune-driven neuroinflammation, demyelination, and failed remyelination. Carnosine is a histidine-containing dipeptide (HCD) with pluripotent homeostatic properties that is able to improve outcomes in an animal MS model (EAE) when supplied exogenously. To uncover if endogenous carnosine is involved in, and protects against, MS-related neuroinflammation, demyelination or remyelination failure, we here studied the HCD-synthesizing enzyme carnosine synthase (CARNS1) in human MS lesions and two preclinical mouse MS models (EAE, cuprizone). We demonstrate that due to its presence in oligodendrocytes, CARNS1 expression is diminished in demyelinated MS lesions and mouse models mimicking demyelination/inflammation, but returns upon remyelination. Carns1-KO mice that are devoid of endogenous HCDs display exaggerated neuroinflammation and clinical symptoms during EAE, which could be partially rescued by exogenous carnosine treatment. Worsening of the disease appears to be driven by a central, not peripheral immune-modulatory, mechanism possibly linked to impaired clearance of the reactive carbonyl acrolein in Carns1-KO mice. In contrast, CARNS1 is not required for normal oligodendrocyte precursor cell differentiation and (re)myelin to occur, and neither endogenous nor exogenous HCDs protect against cuprizone-induced demyelination. In conclusion, the loss of CARNS1 from demyelinated MS lesions can aggravate disease progression through weakening the endogenous protection against neuroinflammation.
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Carnosina , Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Humanos , Ratones , Animales , Esclerosis Múltiple/tratamiento farmacológico , Cuprizona/efectos adversos , Cuprizona/metabolismo , Carnosina/efectos adversos , Carnosina/metabolismo , Enfermedades Neuroinflamatorias , Vaina de Mielina/patología , Oligodendroglía/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patologíaRESUMEN
Insufficient immune tolerance during pregnancy is associated with pathological conditions such as preeclampsia (PE). Soluble fms-like tyrosine kinase-1 (sFLT1), which exerts a role in the late stage of PE, has shown its beneficial anti-inflammatory effects in inflammation-associated diseases. Macrophage migration inhibitory factor (MIF) was reported to upregulate sFLT1 production in experimental congenital diaphragmatic hernia. However, the placental sFLT1 expression in early uncomplicated pregnancy and whether MIF can regulate sFLT1 expression in uncomplicated and preeclamptic pregnancy are unclear. We collected first-trimester placentas and term placentas from uncomplicated and preeclamptic pregnancies to investigate sFLT1 and MIF expression in vivo. Primary cytotrophoblasts (CTBs) and a human trophoblast cell line (Bewo) were used to study the regulation of MIF on sFLT1 expression in vitro. In placentas from first-trimester pregnancy, we observed a high expression of sFLT1, specifically in extravillous trophoblasts (EVTs) and syncytiotrophoblast (STB) cells. MIF mRNA levels strongly correlated with sFLT1 expression in term placentas from preeclamptic pregnancies. In in vitro experiments, sFLT1 and MIF levels increased significantly in CTBs during their differentiation to EVTs and STBs, and MIF inhibitor (ISO-1) significantly reduced sFLT1 expression in a dose-dependent manner during this process. sFLT1 showed significant upregulation with increasing doses of MIF in Bewo cells. Our results show that sFLT1 is highly expressed at the maternal-fetal interface during early pregnancy and that MIF can increase sFLT1 expression in early uncomplicated pregnancy and PE, which suggests that sFLT1 plays an essential role in the modulation of inflammation in pregnancy.
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Factores Inhibidores de la Migración de Macrófagos , Preeclampsia , Embarazo , Femenino , Humanos , Preeclampsia/metabolismo , Placenta/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Trofoblastos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Inflamación/metabolismo , Oxidorreductasas Intramoleculares/metabolismoRESUMEN
Leptin is a hormone that plays a key role in controlling food intake and energy homeostasis. Skeletal muscle is an important target for leptin and recent studies have shown that leptin deficiency may lead to muscular atrophy. However, leptin deficiency-induced structural changes in muscles are poorly understood. The zebrafish has emerged as an excellent model organism for studies of vertebrate diseases and hormone response mechanisms. In this study, we explored ex-vivo magnetic resonance microimaging (µMRI) methods to non-invasively assess muscle wasting in leptin-deficient (lepb-/-) zebrafish model. The fat mapping performed by using chemical shift selective imaging shows significant fat infiltration in muscles of lepb-/- zebrafish compared to control zebrafish. T2 relaxation measurements show considerably longer T2 values in the muscle of lepb-/- zebrafish. Multiexponential T2 analysis detected a significantly higher value and magnitude of long T2 component in the muscles of lepb-/- as compared to control zebrafish. For further zooming into the microstructural changes, we applied diffusion-weighted MRI. The results show a significant decrease in the apparent diffusion coefficient indicating increased constraints of molecular movements within the muscle regions of lepb-/- zebrafish. The use of the phasor transformation for the separation of diffusion-weighted decay signals showed a bi-component diffusion system which allows us to estimate each fraction on a voxel-wise basis. A substantial difference was found between the ratio of two components in lepb-/- and control zebrafish muscles, indicating alterations in diffusion behavior associated with the tissue microstructural changes in muscles of lepb-/- zebrafish as compared to control zebrafish. Taken together, our results demonstrate that the muscles of lepb-/- zebrafish undergo significant fat infiltration and microstructural changes leading to muscle wasting. This study also demonstrates that µMRI provides excellent means to non-invasively study the microstructural changes in the muscles of the zebrafish model.
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Leptina , Pez Cebra , Animales , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Músculo Esquelético/diagnóstico por imagen , Imagen de Difusión por Resonancia Magnética/métodos , Atrofia MuscularRESUMEN
AIMS: Simple Bone Cysts (SBCs) predominantly occur in long bones and 59% harbour NFATC2 rearrangements. Jaw SBC is rare and was previously referred to as traumatic bone cyst. It can rarely occur in association with cemento-osseous dysplasia (COD). To determine whether jaw SBCs represent the same entity as SBC of the long bones, or if they have a different molecular signature, we collected 48 jaw SBC cases of 47 patients to assess NFATC2 rearrangement. METHODS AND RESULTS: Out of the 48 cases, 36 could be used for fluorescence in-situ hybridization (FISH), of which nine (two of which associated with COD) were successful using an NFATC2 split probe. The remaining cases failed to show adequate FISH signals. All nine cases lacked NFATC2 rearrangement and five of these showed no detectable gene fusions using Archer FusionPlex. CONCLUSION: In our study, NFATC2 rearrangement is absent in solitary jaw SBC (n = 7) and COD-associated SBC (n = 2). Our findings suggest that SBC presenting in the jaw is molecularly different from SBC in long bones. Future molecular studies may confirm the absence of clonal molecular aberrations in SBC of the jaw which would support a non-neoplastic, reactive origin.
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Quistes Óseos , Factores de Transcripción NFATC , Tumores Odontogénicos , Humanos , Quistes Óseos/genética , Tumores Odontogénicos/genética , Factores de Transcripción NFATC/genéticaRESUMEN
Psammomatoid ossifying fibroma (PsOF), also known as juvenile PsOF, is a benign fibro-osseous neoplasm predominantly affecting the extragnathic bones, particularly the frontal and ethmoid bones, with a preference for adolescents and young adults. The clinical and morphologic features of PsOF may overlap with those of other fibro-osseous lesions, and additional molecular markers would help increase diagnostic accuracy. Because identical chromosomal breakpoints at bands Xq26 and 2q33 have been described in 3 cases of PsOF located in the orbita, we aimed to identify the exact genes involved in these chromosomal breakpoints and determine their frequency in PsOF using transcriptome sequencing and fluorescence in situ hybridization (FISH). We performed whole RNA transcriptome sequencing on frozen tissue in 2 PsOF index cases and identified a fusion transcript involving SATB2, located on chromosome 2q33.1, and AL513487.1, located on chromosome Xq26, in one of the cases. The fusion was validated using reverse transcription (RT)-PCR and SATB2 FISH. The fusion lead to a truncated protein product losing most of the functional domains. Subsequently, we analyzed an additional 24 juvenile PsOFs, 8 juvenile trabecular ossifying fibromas (JTOFs), and 11 cemento-ossifying fibromas (COFs) for SATB2 using FISH and found evidence of SATB2 gene rearrangements in 58% (7 of 12) of the evaluable PsOF cases but not in any of the evaluable JTOF (n = 7) and COF (n = 7) cases. A combination of SATB2 immunofluorescence and a 2-color SATB2 FISH in our index case revealed that most tumor cells harboring the rearrangement lacked SATB2 expression. Using immunohistochemistry, 65% of PsOF, 100% of JTOF, and 100% of COF cases showed moderate or strong staining for SATB2. In these cases, we observed a mosaic pattern of expression with >25% of the spindle cells in between the bone matrix, with osteoblasts and osteocytes being positive for SATB2. Interestingly, 35% (8 of 23) of PsOFs, in contrast to JTOFs and COFs, showed SATB2 expression in <5% of cells. To our knowledge, this is the first report that shows the involvement of SATB2 in the development of a neoplastic lesion. In this study, we have showed that SATB2 rearrangement is a recurrent molecular alteration that appears to be highly specific for PsOF. Our findings support that PsOF is not only morphologically and clinically but also genetically distinct from JTOF and COF.
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Neoplasias Óseas , Fibroma Osificante , Proteínas de Unión a la Región de Fijación a la Matriz , Humanos , Fibroma Osificante/genética , Hibridación Fluorescente in Situ , Neoplasias Óseas/genética , Inmunohistoquímica , Reordenamiento Génico , Factores de Transcripción/genética , Proteínas de Unión a la Región de Fijación a la Matriz/genéticaRESUMEN
There are no validated housekeeping genes in induced pluripotent stem cells (iPSC) and derived endothelial iPSC (iPSC-EC). Thus a comparison of gene expression levels is less reliable, especially during drug treatments. Here, we utilized transcriptome sequencing data of iPSC and iPSC-EC with or without CRISPR-Cas9 induced translocation to identify a panel of 15 candidate housekeeping genes. For comparison, five commonly used housekeeping genes (B2M, GAPDH, GUSB, HMBS, and HPRT1) were included in the study. The panel of 20 candidate genes were investigated for their stability as reference genes. This panel was analyzed and ranked based on stability using five algorithms, delta-Ct, bestkeeper, geNorm, Normfinder, and Reffinder. Based on the comprehensive ranking of Reffinder, the stability of the top two genes-RPL36AL and TMBIM6, and the bottom two genes-UBA1 and B2M, were further studied in iPSC-EC with and without genetic manipulation, and after treatment with telatinib. Using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), it was shown that gene expression of the top two housekeeping genes, RPL36AL and TMBIM6, remained stable during drug treatment. We identified a panel of housekeeping genes that could be utilized in various conditions using iPSC and iPSC-derived endothelial cells as well as genetically modified iPSC for drug treatment.
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Genes Esenciales , Células Madre Pluripotentes Inducidas , Células Endoteliales , Perfilación de la Expresión Génica , Reacción en Cadena en Tiempo Real de la Polimerasa , Estándares de Referencia , TranscriptomaRESUMEN
(1) Background: Soluble Fms-like tyrosine kinase 1 (sFLT1) is an endogenous VEGF inhibitor. sFLT1 has been described as an anti-inflammatory treatment for diabetic nephropathy and heart fibrosis. However, sFLT1 has also been related to peritubular capillary (PTC) loss, which promotes fibrogenesis. Here, we studied whether transfection with sFlt1 aggravates experimental AKI-to-CKD transition and whether sFLT1 is increased in human kidney fibrosis. (2) Methods: Mice were transfected via electroporation with sFlt1. After confirming transfection efficacy, mice underwent unilateral ischemia/reperfusion injury (IRI) and were sacrificed 28 days later. Kidney histology and RNA were analyzed to study renal fibrosis, PTC damage and inflammation. Renal sFLT1 mRNA expression was measured in CKD biopsies and control kidney tissue. (3) Results: sFlt1 transfection did not aggravate renal fibrosis, PTC loss or macrophage recruitment in IRI mice. In contrast, higher transfection efficiency was correlated with reduced expression of pro-fibrotic and pro-inflammatory markers. In the human samples, sFLT1 mRNA levels were similar in CKD and control kidneys and were not correlated with interstitial fibrosis or PTC loss. (4) Conclusion: As we previously found that sFLT1 has therapeutic potential in diabetic nephropathy, our findings indicate that sFLT1 can be administered at a dose that is therapeutically effective in reducing inflammation, without promoting maladaptive kidney damage.
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Lesión Renal Aguda , Nefropatías Diabéticas , Insuficiencia Renal Crónica , Daño por Reperfusión , Lesión Renal Aguda/metabolismo , Inhibidores de la Angiogénesis , Animales , Fibrosis , Humanos , Inflamación , Ratones , ARN Mensajero , Daño por Reperfusión/metabolismo , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
OBJECTIVES: We aimed to determine the presence, amount and origin of microchimerism in peripheral blood of pregnant and non-pregnant parous women with systemic lupus erythematosus (SLE) as compared to control subjects. METHODS: We performed a comparative study in which peripheral blood was drawn from eleven female non-pregnant SLE-patients and 22 control subjects, and from six pregnant SLE-patients and eleven control subjects during gestation and up to six months postpartum. Quantitative PCR for insertion-deletion polymorphisms and null alleles was used to detect microchimerism in peripheral blood mononuclear cells and granulocytes. RESULTS: Microchimerism was detected more often in non-pregnant SLE-patients than control subjects (54.4% vs. 13.6%, respectively; p=0.03). When present, the median total number of foetal chimeric cells was 5 gEq/106 in patients and 2.5gEq/106 in control subjects (p=0.048). Microchimerism was mostly foetal in origin; maternal microchimerism was detected in one patient and one control subject. In control subjects, microchimerism was always derived from only one source whereas in 50% of patients it originated from multiple sources. The pregnant patients had a significantly higher median number of foetal chimeric cells in the granulocyte fraction just after delivery than control subjects (7.5 gEq/106 vs. 0 gEq/106, respectively; p=0.02). CONCLUSIONS: Just after delivery, SLE-patients had more microchimerism than control subjects. Three months post-partum, microchimerism was no longer detectable, only to reappear many years after the last pregnancy, more often and at higher levels in SLE-patients than in control subjects. This suggests that these chimeric cells may originate from non-circulating foetal chimeric stem cells.
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Lupus Eritematoso Sistémico , Complicaciones del Embarazo , Embarazo , Humanos , Femenino , Quimerismo , Leucocitos Mononucleares , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Vascular endothelial growth factor antagonism with angiogenesis inhibitors in cancer patients induces a 'preeclampsia-like' syndrome including hypertension, proteinuria and elevated endothelin (ET)-1. Cyclo-oxygenase (COX) inhibition with aspirin is known to prevent the onset of preeclampsia in high-risk patients. In the present study, we hypothesised that treatment with aspirin would prevent the development of angiogenesis inhibitor-induced hypertension and kidney damage. Our aims were to compare the effects of low-dose (COX-1 inhibition) and high-dose (dual COX-1 and COX-2 inhibition) aspirin on blood pressure, vascular function, oxidative stress, ET-1 and prostanoid levels and kidney damage during angiogenesis-inhibitor therapy in rodents. To this end, Wistar Kyoto rats were treated with vehicle, angiogenesis inhibitor (sunitinib) alone or in combination with low- or high-dose aspirin for 8 days (n=5-7/group). Our results demonstrated that prostacyclin (PGI2) and ET-1 were increased during angiogenesis-inhibitor therapy, while thromboxane (TXA2) was unchanged. Both low- and high-dose aspirin blunted angiogenesis inhibitor-induced hypertension and vascular superoxide production to a similar extent, whereas only high-dose aspirin prevented albuminuria. While circulating TXA2 and prostaglandin F2α levels were reduced by both low- and high-dose aspirin, circulating and urinary levels PGI2 were only reduced by high-dose aspirin. Lastly, treatment with aspirin did not significantly affect ET-1 or vascular function. Collectively our findings suggest that prostanoids contribute to the development of angiogenesis inhibitor-induced hypertension and renal damage and that targeting the prostanoid pathway could be an effective strategy to mitigate the unwanted cardiovascular and renal toxicities associated with angiogenesis inhibitors.
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Hipertensión , Preeclampsia , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Aspirina/farmacología , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Endotelina-1/metabolismo , Epoprostenol/metabolismo , Epoprostenol/farmacología , Epoprostenol/uso terapéutico , Femenino , Humanos , Hipertensión/inducido químicamente , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismo , Riñón/metabolismo , Preeclampsia/inducido químicamente , Preeclampsia/tratamiento farmacológico , Preeclampsia/metabolismo , Embarazo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Ratas , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
In this study, the effect of heterozygous germline mutations in the heparan sulfate (HS) glycosaminoglycan chain co-polymerases EXT1 and EXT2 on glomerular barrier function and the endothelial glycocalyx in humans is investigated. Heparan sulfate (HS) glycosaminoglycans are deemed essential to the glomerular filtration barrier, including the glomerular endothelial glycocalyx. Animal studies have shown that loss of HS results in a thinner glycocalyx. Also, decreased glomerular HS expression is observed in various proteinuric renal diseases in humans. A case report of a patient with an EXT1 mutation indicated that this could result in a specific renal phenotype. This patient suffered from multiple osteochondromas, an autosomal dominant disease caused by mono-allelic germline mutations in the EXT1 or EXT2 gene. These studies imply that HS is indeed essential to the glomerular filtration barrier. However, loss of HS did not lead to proteinuria in various animal models. We demonstrate that multiple osteochondroma patients do not have more microalbuminuria or altered glycocalyx properties compared to age-matched controls (n = 19). A search for all Dutch patients registered with both osteochondroma and kidney biopsy (n = 39) showed that an EXT1 or EXT2 mutation does not necessarily lead to specific glomerular morphological phenotypic changes. In conclusion, this study shows that a heterozygous mutation in the HS backbone elongating enzymes EXT1 and EXT2 in humans does not result in (micro)albuminuria, a specific renal phenotype or changes to the endothelial glycocalyx, adding to the growing knowledge on the role of EXT1 and EXT2 genes in pathophysiology.
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Barrera de Filtración Glomerular , Glicocálix , N-Acetilglucosaminiltransferasas , Barrera de Filtración Glomerular/metabolismo , Glicocálix/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Mutación , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismoRESUMEN
Chronic kidney disease (CKD) is a slow-developing, progressive deterioration of renal function. The final common pathway in the pathophysiology of CKD involves glomerular sclerosis, tubular atrophy and interstitial fibrosis. Transforming growth factor-beta (TGF-ß) stimulates the differentiation of fibroblasts towards myofibroblasts and the production of extracellular matrix (ECM) molecules, and thereby interstitial fibrosis. It has been shown that endoglin (ENG, CD105), primarily expressed in endothelial cells and fibroblasts, can function as a co-receptor of TGF signaling. In several human organs, endoglin tends to be upregulated when chronic damage and fibrosis is present. We hypothesize that endoglin is upregulated in renal interstitial fibrosis and plays a role in the progression of CKD. We first measured renal endoglin expression in biopsy samples obtained from patients with different types of CKD, i.e., IgA nephropathy, focal segmental glomerulosclerosis (FSGS), diabetic nephropathy (DN) and patients with chronic allograft dysfunction (CAD). We showed that endoglin is upregulated in CAD patients (p < 0.001) and patients with DN (p < 0.05), compared to control kidneys. Furthermore, the amount of interstitial endoglin expression correlated with eGFR (p < 0.001) and the amount of interstitial fibrosis (p < 0.001), independent of the diagnosis of the biopsies. Finally, we investigated in vitro the effect of endoglin overexpression in TGF-ß stimulated human kidney fibroblasts. Overexpression of endoglin resulted in an enhanced ACTA2, CCN2 and SERPINE1 mRNA response (p < 0.05). It also increased the mRNA and protein upregulation of the ECM components collagen type I (COL1A1) and fibronectin (FN1) (p < 0.05). Our results suggest that endoglin is an important mediator in the final common pathway of CKD and could be used as a possible new therapeutic target to counteract the progression towards end-stage renal disease (ESRD).
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Nefropatías Diabéticas , Endoglina , Fallo Renal Crónico , Insuficiencia Renal Crónica , Humanos , Nefropatías Diabéticas/metabolismo , Endoglina/genética , Endoglina/metabolismo , Células Endoteliales/metabolismo , Fibrosis , Riñón/metabolismo , Fallo Renal Crónico/patología , Receptores de Factores de Crecimiento/metabolismo , Insuficiencia Renal Crónica/metabolismo , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Pre-eclampsia has a major impact on renal function as shown by the development of proteinuria and podocyturia. How the systemic, soluble Fms-like tyrosine kinase-1 (sFlt-1)-driven inhibition of vascular endothelial growth factor (VEGF) activity detected in pre-eclampsia directly affects renal function remains unknown. The aim of the study was to clarify whether a non-canonical, renal-centred escape from VEGF inhibition in the case of pre-eclamptic pregnancy might have a direct impact on renal function. METHODS: We evaluated plasma and urinary VEGF and placental growth factor (PlGF), plasma sFlt-1 and carbonic anhydrase IX (CAIX), albuminuria and podocyturia in 18 women with uncomplicated pregnancy, 21 with pre-eclampsia and 18 non-pregnant. The three groups were matched for age and the pregnant groups also for gestational age at enrolment. RESULTS: Plasma VEGF was reduced in uncomplicated (P = 0.001) and pre-eclamptic (P = 0.0003) pregnancies when compared with controls. In uncomplicated pregnancy, the dysfunction was balanced by an increase (P = 0.009) of plasma PlGF. Increased (P = 0.0001) plasma CAIX in pre-eclampsia was in line with hypoxia. Pre-eclampsia resulted in a paradoxical increase (P = 0.0004) of urinary excretion of VEGF. Urinary concentrations of VEGF and podocytes were correlated to each other (r2 = 0.48, P < 0.0005) but also to plasma sFlt-1 (r2 = 0.56, P < 0.0001 and r2 = 0.23, P = 0.03, respectively). CONCLUSIONS: In the case of pre-eclampsia, the systemic VEGF inhibition leads the kidney, possibly the podocyte, to increase the VEGF synthesis. The mechanisms leading to local VEGF overproduction or the overproduced VEGF itself are reasonably involved in the pathogenesis of podocyturia and, as a consequence, renal dysfunction in pre-eclampsia.
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Enfermedades Renales , Preeclampsia , Biomarcadores , Femenino , Humanos , Factor de Crecimiento Placentario , Preeclampsia/etiología , Preeclampsia/patología , Embarazo , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial VascularRESUMEN
Cystinosis is a rare, incurable, autosomal recessive disease caused by mutations in the CTNS gene. This gene encodes the lysosomal cystine transporter cystinosin, leading to lysosomal cystine accumulation in all cells of the body, with kidneys being the first affected organs. The current treatment with cysteamine decreases cystine accumulation, but does not reverse the proximal tubular dysfunction, glomerular injury or loss of renal function. In our previous study, we have developed a zebrafish model of cystinosis through a nonsense mutation in the CTNS gene and have shown that zebrafish larvae recapitulate the kidney phenotype described in humans. In the current study, we characterized the adult cystinosis zebrafish model and evaluated the long-term effects of the disease on kidney and extra renal organs through biochemical, histological, fertility and locomotor activity studies. We found that the adult cystinosis zebrafish presents cystine accumulation in various organs, altered kidney morphology, impaired skin pigmentation, decreased fertility, altered locomotor activity and ocular anomalies. Overall, our data indicate that the adult cystinosis zebrafish model reproduces several human phenotypes of cystinosis and may be useful for studying pathophysiology and long-term effects of novel therapies.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Cistina/metabolismo , Cistinosis/patología , Modelos Animales de Enfermedad , Riñón/patología , Mutación , Proteínas de Pez Cebra/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Cistinosis/etiología , Humanos , Riñón/metabolismo , Fenotipo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a disease that causes thrombocytopenia and a risk of bleeding in the (unborn) child that result from maternal alloantibodies directed against fetal, paternally inherited, human platelet antigens (HPA). It is hypothesized that these alloantibodies can also bind to the placenta, causing placental damage. This study aims to explore signs of antibody-mediated placental damage in FNAIT. We performed a retrospective study that included pregnant women, their newborns, and placentas. It comprised 23 FNAIT cases, of which nine were newly diagnosed (14 samples) and 14 were antenatally treated with intravenous immune globulins (IVIg) (21 samples), and 20 controls, of which 10 had anti-HLA-class I antibodies. Clinical information was collected from medical records. Placental samples were stained for complement activation markers (C1q, C4d, SC5b-9, and mannose-binding lectin) using immunohistochemistry. Histopathology was examined according to the Amsterdam criteria. A higher degree of C4d deposition was present in the newly diagnosed FNAIT cases (10/14 samples), as compared to the IVIg-treated FNAIT cases (2/21 samples, p = 0.002) and anti-HLA-negative controls (3/20 samples, p = 0.006). A histopathological examination showed delayed maturation in four (44%) placentas in the newly diagnosed FNAIT cases, five (36%) in the IVIg-treated FNAIT cases, and one in the controls (NS). C4d deposition at the syncytiotrophoblast was present in combination with low-grade villitis of unknown etiology in three newly diagnosed FNAIT cases that were born SGA. We conclude that a higher degree of classical pathway-induced complement activation is present in placentas from pregnancies with untreated FNAIT. This may affect placental function and fetal growth.