RESUMEN
We report the case of two siblings with incomplete Donnai-Barrow syndrome (DBS) phenotype carrying three LRP2 variants never associated before with DBS phenotype.
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Pérdida Auditiva Sensorineural , Hernias Diafragmáticas Congénitas , Miopía , Humanos , Niño , Pérdida Auditiva Sensorineural/genética , Miopía/genética , Hernias Diafragmáticas Congénitas/diagnóstico , Proteinuria/diagnóstico , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genéticaRESUMEN
Dent disease (DD1) is a rare tubulopathy caused by mutations in the CLCN5 gene. Glomerulosclerosis was recently reported in DD1 patients and ClC-5 protein was shown to be expressed in human podocytes. Nephrin and actin cytoskeleton play a key role for podocyte functions and podocyte endocytosis seems to be crucial for slit diaphragm regulation. The aim of this study was to analyze whether ClC-5 loss in podocytes might be a direct consequence of the glomerular damage in DD1 patients. Three DD1 kidney biopsies presenting focal global glomerulosclerosis and four control biopsies were analyzed by immunofluorescence (IF) for nephrin and podocalyxin, and by immunohistochemistry (IHC) for ClC-5. ClC-5 resulted as down-regulated in DD1 vs. control (CTRL) biopsies in both tubular and glomerular compartments (p < 0.01). A significant down-regulation of nephrin (p < 0.01) in DD1 vs. CTRL was demonstrated. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Caspase9) gene editing of CLCN5 in conditionally immortalized human podocytes was used to obtain clones with the stop codon mutation p.(R34Efs*14). We showed that ClC-5 and nephrin expression, analyzed by quantitative Reverse Transcription/Polymerase Chain Reaction (qRT/PCR) and In-Cell Western (ICW), was significantly downregulated in mutant clones compared to the wild type ones. In addition, F-actin staining with fluorescent phalloidin revealed actin derangements. Our results indicate that ClC-5 loss might alter podocyte function either through cytoskeleton disorganization or through impairment of nephrin recycling.
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Canales de Cloruro , Enfermedad de Dent , Glomeruloesclerosis Focal y Segmentaria , Podocitos , Humanos , Actinas/genética , Actinas/metabolismo , Enfermedad de Dent/genética , Enfermedad de Dent/patología , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomérulos Renales/metabolismo , Podocitos/metabolismo , Canales de Cloruro/metabolismoRESUMEN
Dent disease (DD), an X-linked renal tubulopathy, is mainly caused by loss-of-function mutations in CLCN5 (DD1) and OCRL genes. CLCN5 encodes the ClC-5 antiporter that in proximal tubules (PT) participates in the receptor-mediated endocytosis of low molecular weight proteins. Few studies have analyzed the PT expression of ClC-5 and of megalin and cubilin receptors in DD1 kidney biopsies. About 25% of DD cases lack mutations in either CLCN5 or OCRL genes (DD3), and no other disease genes have been discovered so far. Sanger sequencing was used for CLCN5 gene analysis in 158 unrelated males clinically suspected of having DD. The tubular expression of ClC-5, megalin, and cubilin was assessed by immunolabeling in 10 DD1 kidney biopsies. Whole exome sequencing (WES) was performed in eight DD3 patients. Twenty-three novel CLCN5 mutations were identified. ClC-5, megalin, and cubilin were significantly lower in DD1 than in control biopsies. The tubular expression of ClC-5 when detected was irrespective of the type of mutation. In four DD3 patients, WES revealed 12 potentially pathogenic variants in three novel genes (SLC17A1, SLC9A3, and PDZK1), and in three genes known to be associated with monogenic forms of renal proximal tubulopathies (SLC3A, LRP2, and CUBN). The supposed third Dent disease-causing gene was not discovered.
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Canales de Cloruro/genética , Enfermedad de Dent/genética , Enfermedad de Dent/patología , Predisposición Genética a la Enfermedad , Enfermedades Renales/genética , Enfermedades Renales/patología , Mutación , Biomarcadores , Biopsia , Análisis Mutacional de ADN , Estudios de Asociación Genética , Humanos , Inmunohistoquímica , Secuenciación del ExomaRESUMEN
Apoptotic cell death is usually a response to the cell's microenvironment. In the kidney, apoptosis contributes to parenchymal cell loss in the course of acute and chronic renal injury, but does not trigger an inflammatory response. What distinguishes necrosis from apoptosis is the rupture of the plasma membrane, so necrotic cell death is accompanied by the release of unprocessed intracellular content, including cellular organelles, which are highly immunogenic proteins. The relative contribution of apoptosis and necrosis to injury varies, depending on the severity of the insult. Regulated cell death may result from immunologically silent apoptosis or from immunogenic necrosis. Recent advances have enhanced the most revolutionary concept of regulated necrosis. Several modalities of regulated necrosis have been described, such as necroptosis, ferroptosis, pyroptosis, and mitochondrial permeability transition-dependent regulated necrosis. We review the different modalities of apoptosis, necrosis, and regulated necrosis in kidney injury, focusing particularly on evidence implicating cell death in ectopic renal calcification. We also review the evidence for the role of cell death in kidney injury, which may pave the way for new therapeutic opportunities.
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Lesión Renal Aguda/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Calcinosis/metabolismo , Células Epiteliales/metabolismo , Riñón/metabolismo , Necrosis/metabolismo , Daño por Reperfusión/metabolismo , Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/clasificación , Proteínas Reguladoras de la Apoptosis/metabolismo , Calcinosis/genética , Calcinosis/patología , Calcinosis/prevención & control , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Ferroptosis/efectos de los fármacos , Ferroptosis/genética , Regulación de la Expresión Génica , Humanos , Muerte Celular Inmunogénica/efectos de los fármacos , Muerte Celular Inmunogénica/genética , Riñón/efectos de los fármacos , Riñón/patología , Necrosis por Permeabilidad de la Transmembrana Mitocondrial/efectos de los fármacos , Necrosis por Permeabilidad de la Transmembrana Mitocondrial/genética , Necroptosis/efectos de los fármacos , Necroptosis/genética , Necrosis/genética , Necrosis/patología , Necrosis/prevención & control , Sustancias Protectoras/farmacología , Piroptosis/efectos de los fármacos , Piroptosis/genética , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/genética , Daño por Reperfusión/patologíaRESUMEN
Medullary nephrocalcinosis is a hallmark of medullary sponge kidney (MSK). We had the opportunity to study a spontaneous calcification process in vitro by utilizing the renal cells of a patient with MSK who was heterozygous for the c.-27 + 18G>A variant in the GDNF gene encoding glial cell-derived neurotrophic factor. The cells were obtained by collagenase digestion of papillary tissues from the MSK patient and from two patients who had no MSK or nephrocalcinosis. These cells were typed by immunocytochemistry, and the presence of mineral deposits was studied using von Kossa staining, scanning electron microscopy analysis and an ALP assay. Osteoblastic lineage markers were studied using immunocytochemistry and RT-PCR. Staminality markers were also analysed using flow cytometry, magnetic cell separation technology, immunocytochemistry and RT-PCR. Starting from p2, MSK and control cells formed nodules with a behaviour similar to that of calcifying pericytes; however, Ca2PO4 was only found in the MSK cultures. The MSK cells had morphologies and immunophenotypes resembling those of pericytes or stromal stem cells and were positive for vimentin, ZO1, αSMA and CD146. In addition, the MSK cells expressed osteocalcin and osteonectin, indicating an osteoblast-like phenotype. In contrast to the control cells, GDNF was down-regulated in the MSK cells. Stable GDNF knockdown was established in the HK2 cell line and was found to promote Ca2PO4 deposition when the cells were incubated with calcifying medium by regulating the osteonectin/osteopontin ratio in favour of osteonectin. Our data indicate that the human papilla may be a perivascular niche in which pericyte/stromal-like cells can undergo osteogenic differentiation under particular conditions and suggest that GDNF down-regulation may have influenced the observed phenomenon.
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Calcinosis , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Riñón Esponjoso Medular/genética , Mutación , Actinas/metabolismo , Anciano , Antígeno CD146/metabolismo , Calcificación Fisiológica , Línea Celular , Células Cultivadas , Femenino , Humanos , Inmunohistoquímica , Riñón/metabolismo , Riñón/patología , Riñón/ultraestructura , Riñón Esponjoso Medular/metabolismo , Riñón Esponjoso Medular/patología , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Músculo Liso/química , Osteonectina/genética , Osteonectina/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Cultivo Primario de Células , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vimentina/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
BACKGROUND: Parietal epithelial cells are a heterogeneous population of cells located on Bowman's capsule. These cells are known to internalize albumin with a still undetermined mechanism, although albumin has been shown to induce phenotypic changes in parietal epithelial cells. Proximal tubular cells are the main actors in albumin handling via the macromolecular complex composed by ClC-5, megalin, and cubilin. This study investigated the role of ClC-5, megalin, and cubilin in the parietal epithelial cells of kidney biopsies from proteinuric lupus nephritis patients and control subjects and identified phenotypical changes occurring in the pathological milieu. METHODS: Immunohistochemistry and immunofluorescence analyses for ClC-5, megalin, cubilin, ANXA3, podocalyxin, CD24, CD44, HSA, and LTA marker were performed on 23 kidney biopsies from patients with Lupus Nephritis and 9 control biopsies (obtained from nephrectomies for renal cancer). RESULTS: Two sub-populations of hypertrophic parietal epithelial cells ANXA3+/Podocalyxin-/CD44-, both expressing ClC-5, megalin, and cubilin and located at the tubular pole, were identified and characterized: the first one, CD24+/HSA-/LTA- had characteristics of human adult parietal epithelial multipotent progenitors, the second one, CD24-/LTA+/HSA+ committed to become phenotypically proximal tubular cells. The number of glomeruli presenting hypertrophic parietal epithelial cells positive for ClC-5, megalin, and cubilin were significantly higher in lupus nephritis patients than in controls. CONCLUSIONS: Our results may provide further insight into the role of hypertrophic parietal epithelial cells located at the tubular pole and their possible involvement in protein endocytosis in lupus nephritis patients. These data also suggest that the presence of hypertrophic parietal epithelial cells in Bowman's capsule represents a potential resource for responding to protein overload observed in other glomerulonephritis.
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Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad , Nefritis Lúpica , Humanos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Túbulos Renales Proximales , Proteinuria/etiología , Albúminas/metabolismo , Células Epiteliales/metabolismoRESUMEN
A number of glycosaminoglycan (GAG) species related to heparin, dermatan sulfate (DeS) and chondroitin sulfate were tested for their ability to interfere with the physiological expression and/or pathological overexpression of the TGF-ß1 gene. The influence of the molecular weight, molecular weight distribution, degree of sulfation and location of the sulfate groups was examined in an attempt to unveil fine relationships between structure and activity. The nature of the polysaccharide plays a major part, heparins proving able to inhibit both basal and stimulated TGF-ß1 gene expression, DeSs being essentially inactive and chondroitin sulfates only inhibiting stimulated TGF-ß1 gene expression. Within this frame, the particular physical and chemical properties of some GAGs appear to further modulate TGF-ß1 gene response. Judging from our investigation, chondroitin sulfates seem the most promising for potential pharmacological applications in disorders characterized by fibrogenic TGF-ß1 overexpression.
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Glicosaminoglicanos/farmacología , Células Mesangiales/efectos de los fármacos , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Animales , Conformación de Carbohidratos , Células Cultivadas , Glicosaminoglicanos/química , Células Mesangiales/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Relación Estructura-Actividad , Porcinos , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Fabry disease is an X-linked disorder due to mutations in α-galactosidase A, resulting in the accumulation of enzyme substrates and cell malfunction. Kidney involvement is frequent, affecting all native kidney cell types. Podocyte damage results in proteinuria and chronic kidney disease. End-stage kidney disease is the rule in middle-aged males and some females with the classic phenotype. In podocytes and kidney proximal tubular cells, megalin is one of the molecules involved in enzyme replacement therapy (ERT) cellular absorption. After podocyte damage, podocin concentration is decreased and contributes to progressive proteinuria. We report in a male and a female patient the decreased expression of megalin, cubilin, ClC-5 and podocin compared to controls and chronic kidney disease (CKD) biopsies. Moreover, the decrease in ClC-5, a molecule engaged in endosomal-lysosomal acidification, could also affect ERT. These findings may partially explain some of the dysfunctions described in Fabry nephropathy and could highlight possible alterations in the pharmacokinetics of the delivered enzyme.
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Enfermedad de Fabry , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad , Canales de Cloruro , Regulación hacia Abajo , Terapia de Reemplazo Enzimático , Enfermedad de Fabry/diagnóstico , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/genética , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Proteínas de la Membrana , Persona de Mediana Edad , Receptores de Superficie CelularRESUMEN
Dent disease is a rare X-linked renal tubulopathy due to CLCN5 and OCRL (DD2) mutations. OCRL mutations also cause Lowe syndrome (LS) involving the eyes, brain and kidney. DD2 is frequently described as a mild form of LS because some patients may present with extra-renal symptoms (ESs). Since DD2 is a rare disease and there are a low number of reported cases, it is still unclear whether it has a clinical picture distinct from LS. We retrospectively analyzed the phenotype and genotype of our cohort of 35 DD2 males and reviewed all published DD2 cases. We analyzed the distribution of mutations along the OCRL gene and evaluated the type and frequency of ES according to the type of mutation and localization in OCRL protein domains. The frequency of patients with at least one ES was 39%. Muscle findings are the most common ES (52%), while ocular findings are less common (11%). Analysis of the distribution of mutations revealed (1) truncating mutations map in the PH and linker domain, while missense mutations map in the 5-phosphatase domain, and only occasionally in the ASH-RhoGAP module; (2) five OCRL mutations cause both DD2 and LS phenotypes; (3) codon 318 is a DD2 mutational hot spot; (4) a correlation was found between the presence of ES and the position of the mutations along OCRL domains. DD2 is distinct from LS. The mutation site and the mutation type largely determine the DD2 phenotype.
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Enfermedades Genéticas Ligadas al Cromosoma X/genética , Pleiotropía Genética/genética , Nefrolitiasis/genética , Síndrome Oculocerebrorrenal/genética , Monoéster Fosfórico Hidrolasas/genética , Adolescente , Variación Biológica Poblacional/genética , Niño , Preescolar , Femenino , Estudios de Asociación Genética , Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico , Enfermedades Genéticas Ligadas al Cromosoma X/epidemiología , Genotipo , Humanos , Riñón/metabolismo , Riñón/patología , Masculino , Mutación Missense/genética , Nefrolitiasis/diagnóstico , Nefrolitiasis/epidemiología , Síndrome Oculocerebrorrenal/diagnóstico , Síndrome Oculocerebrorrenal/epidemiología , FenotipoRESUMEN
ClC-5, the electrogenic chloride/proton exchanger strongly expressed in renal proximal tubules, belongs to the endocytic macromolecular complex responsible for albumin and low-molecular-weight protein uptake. ClC-5 was found to be overexpressed in glomeruli of glomerulonephritis and in cultured human podocytes under albumin overload. The transcriptional regulation of human ClC-5 is not fully understood. Three functional promoters of various strengths and 11 different 5' untranslated region (5'UTR) isoforms of CLCN5 messenger RNA (mRNA) were detected in the human kidney (variants 1-11). The aim of this study was to investigate the expression pattern of CLCN5 5'UTR variants and the CLCN5 common translated region in glomerulonephritis. The 5'UTR ends and the translated region of CLCN5 mRNA were analyzed using quantitative relative real-time PCR or quantitative comparative endpoint PCR with GAPDH as housekeeping gene in 8 normal kidneys and 12 renal biopsies from patients with glomerulonephritis. The expression profile for all variants in normal and glomerulonephritis biopsies was similar, and variant 3 and alternative variant 4 were the most abundantly expressed in both sets. In glomerulonephritis biopsies, isoforms under the control of a weak promoter (variants 4, 6 and 7) showed an increased expression leading to an increase in the CLCN5 translated region, underscoring their importance in kidney pathophysiology. Since weak promoters can be turned on by different stimuli, these data support the hypothesis that proteinuria could be one of the stimuli capable of starting a signaling pathway that induces an increase in CLCN5 transcription.
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Regiones no Traducidas 5'/genética , Canales de Cloruro/genética , Regulación de la Expresión Génica , Glomerulonefritis/genética , Riñón/metabolismo , Anciano , Biopsia , Estudios de Casos y Controles , Canales de Cloruro/metabolismo , Femenino , Perfilación de la Expresión Génica , Glomerulonefritis/patología , Humanos , Masculino , Persona de Mediana Edad , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Proteinuria is a well-known risk factor, not only for renal disorders, but also for several other problems such as cardiovascular diseases and overall mortality. In the kidney, the chloride channel Cl-/H+ exchanger ClC-5 encoded by the CLCN5 gene is actively involved in preventing protein loss. This action becomes evident in patients suffering from the rare proximal tubulopathy Dent disease because they carry a defective ClC-5 due to CLCN5 mutations. In fact, proteinuria is the distinctive clinical sign of Dent disease, and mainly involves the loss of low-molecular-weight proteins. The identification of CLCN5 disease-causing mutations has greatly improved our understanding of ClC-5 function and of the ClC-5-related physiological processes in the kidney. This review outlines current knowledge regarding the CLCN5 gene and its protein product, providing an update on ClC-5 function in tubular and glomerular cells, and focusing on its relationship with proteinuria and Dent disease.
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Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Enfermedad de Dent/genética , Endocitosis , Animales , Canales de Cloruro/química , Enfermedad de Dent/patología , Humanos , Riñón/metabolismo , Mutación/genética , FenotipoRESUMEN
BACKGROUND/AIMS: In kidney transplants, the renin-angiotensin system (RAS) is involved in systemic and local changes that may induce fibrosis. Our aim was to use gene expression and immunohistochemical analysis to investigate the RAS and several factors involved in the fibrogenic cascade in allograft biopsies. METHODS: We considered 43 donor biopsies (T0), 18 biopsies obtained for diagnostic purposes (Td) and 24 protocol biopsies (Tp) taken 2 months after transplantation in patients with stable renal function. Morphometric alpha SMA and TGF beta 1 analysis, and Masson's Trichrome staining were performed. mRNA levels of angiotensinogen, renin, ACE, AT1-R, AT2-R, TGF beta 1, BMP-7, Coll III, fibronectin and alpha SMA were analyzed by real-time RT/PCR. MDRD a year after the transplant was also considered. RESULTS: Significantly higher levels of AT1-R and alpha SMA transcripts were found in Tp than in T0. Regression analysis showed significant TGF beta 1-independent positive correlations between RAS and matrix components in T0 and Tp, but more evident in Tp, where a positive correlation between TGF beta 1 and Masson's Trichrome stained areas was also seen. CONCLUSION: Our results suggest that RAS and TGF beta 1-related fibrogenic loops are activated as early as 2 months after kidney transplantation.
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Perfilación de la Expresión Génica , Trasplante de Riñón/patología , Sistema Renina-Angiotensina/fisiología , Factor de Crecimiento Transformador beta1/genética , Adulto , Biopsia , Fibrosis , Expresión Génica , Humanos , Inmunohistoquímica , Riñón/metabolismo , Riñón/patología , Persona de Mediana Edad , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
BACKGROUND: IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and is characterized by extremely variable clinical and morphological features and outcome. TGF-beta1 has a key role in fibrogenesis and the progression of renal damage. Its production is under genetic control. METHODS: We recruited 105 Italian biopsy-proven IgAN patients for genotyping for the TGF-beta1 C-509T, T869C (COD 10) and G915C (COD 25) polymorphisms; 200 healthy blood donors were used as normal controls. Glomerular and interstitial mRNA levels of TGF-beta1 were assessed by real-time PCR in 34 patients to seek relationships with clinical, renal histopathological features and outcome. RESULTS: The genotype distributions in the IgAN population were not statistically different from the controls. The COD 10 TT genotype was associated with more severe histological damage as assessed by Lee's classification (CC 50%, CT 39.6% and TT 17.2% were graded as mild; CC 35.7%, CT 43.7% and TT 44.8% as moderate, and CC 14.3%, CT 16.7% and TT 37.9% as severe [p=0.0049]) and with severe interstitial infiltrates (CC 10.4%, CT 35.2% and TT 54.2% [p=0.03]). A higher interstitial immunodeposition was observed for TGF-beta1, collagen IV and alpha-SMA in patients with the COD 10 T allele (p=0.045, p=0.049, p=0.032, respectively). The T allele was associated with significantly higher TGF-beta1 mRNA levels in the interstitium (TT+CT vs. CC: 0.52 +/- 0.16 vs. 0.18 +/- 0.10 copies/mL, respectively; p=0.000). The T allele was also associated with higher mRNA levels in glomeruli, though the difference was not statistically significant. Finally, the T allele was significantly associated with a worse prognosis, the end points being reached by 40% of TT+CT and 32% of CC patients (p=0.009). CONCLUSIONS: In primary IgA nephropathy, the T allele of the TGF-beta1 COD 10 C/T polymorphism seems to be associated with more severe histological lesions, higher renal TGF-beta1 mRNA levels and a worse prognosis. This polymorphism seems to be functionally relevant and to have a prognostic impact.
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Glomerulonefritis por IGA/genética , Riñón/metabolismo , Polimorfismo Genético , Factor de Crecimiento Transformador beta1/genética , Adulto , Biopsia , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Glomerulonefritis por IGA/metabolismo , Glomerulonefritis por IGA/patología , Humanos , Italia , Estimación de Kaplan-Meier , Riñón/patología , Masculino , Persona de Mediana Edad , Fenotipo , Pronóstico , Modelos de Riesgos Proporcionales , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Medición de Riesgo , Factores de Riesgo , Índice de Severidad de la Enfermedad , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia Arriba , Adulto JovenRESUMEN
Nephrocalcinosis is a clinicopathological entity characterized by microscopic calcium crystals in the renal parenchyma, within the tubular lumen or in the interstitium. Crystal binding to tubular cells may be the cause underlying nephrocalcinosis and nephrolithiasis. Pathological circumstances, such as acute cortical necrosis, may induce healthy cells to acquire a crystal-binding phenotype. The present study aimed to investigate whether human renal proximal tubular cells (HK-2 cells) can form calcium phosphate deposits under osteogenic conditions, and whether apoptosis and/or osteogenic-like processes are involved in cell calcification. HK-2 cells were cultured in standard or osteogenic medium for 1, 5, and 15 days. Von Kossa staining and ESEM were used to analyze crystal deposition. Apoptosis was investigated, analyzing caspase activation by in-cell Western assay, membrane translocation of phosphotidylserine by annexin V-FITC/propidium iodide staining, and DNA fragmentation by TUNEL assay. qRT/PCR, immunolabeling and cytochemistry were performed to assess osteogenic activation (Runx2, Osteonectin, Osteopontin and ALP), and early genes of apoptosis (BAX, Bcl-2). HK-2 cell mineralization was successfully induced on adding osteogenic medium. Calcium phosphate deposition increased in a time-dependent manner, and calcified cell aggregates exhibited characteristic signs of apoptosis. At 15 days, calcifying HK-2 cells revealed osteogenic markers, such as Runx2, ALP, osteonectin and osteopontin. Monitoring the processes at 1, 5, and 15 days showed apoptosis starting already after 5 days of osteogenic induction, when the first small calcium phosphate crystals began to appear on areas where cell aggregates were in apoptotic conditions. The cell death process proved caspase-dependent. The importance of apoptosis was reinforced by the time-dependent increase in BAX expression, starting from day 1. These findings strongly support the hypothesis that apoptosis triggered HK-2 calcification even before any calcium phosphate crystal deposition or acquisition of an osteogenic phenotype.
RESUMEN
The adult mammalian renal tubular epithelium exists in a relatively quiescent to slowly replicating state, but has great potential for regenerative morphogenesis following severe ischemic or toxic injury. Kidney regeneration and repair occur through three cellular and molecular mechanisms: differentiation of the somatic stem cells, recruitment of circulating stem cells and, more importantly, proliferation/dedifferentiation of mature cells. Dedifferentiation seems to represent a critical step for the recovery of tubular integrity. Dedifferentiation of tubular cells after injury is characterized by the reactivation of a mesenchymal program that is active during nephrogenesis. Epithelial-to-mesenchymal transition (EMT) of renal tubular cells is an extreme manifestation of epithelial cell plasticity. It is now widely recognized as a fundamental process that marks some physiological, such as morphogenesis, as well as pathological events, such as oncogenesis and fibrogenesis. It might be also considered as a key event in the regenerative process of the kidney. Understanding the molecular mechanisms involved in EMT might be useful for designing therapeutic strategies in order to potentiate the innate capacity of the kidney to regenerate.
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Riñón/fisiología , Regeneración/fisiología , Células Madre/fisiología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Diferenciación Celular , Células Epiteliales/citología , Células Epiteliales/fisiología , Humanos , Isquemia/fisiopatología , Riñón/citología , Riñón/lesiones , Riñón/fisiopatología , Túbulos Renales/citología , Túbulos Renales/fisiología , Mamíferos , Valores de Referencia , Circulación Renal/fisiología , Células Madre/citologíaRESUMEN
Randall's plaques are very common in idiopathic calcium-oxalate nephrolithiasis. These papillary plaques have an apatite mineral structure. While these calcium deposits are generally assumed to be secondary to a purely physico-chemical phenomenon, we advance the hypothesis that they form due to a truly ectopic biomineralization in the renal tissue, and that Henle's loop epithelial cells, or pericyte-like interstitial cells, or papillary stem cells differentiating along a bone lineage might be involved.
RESUMEN
Nephrocalcinosis involves the deposition of microscopic crystals in the tubular lumen or interstitium. While the clinical, biochemical, and genetic aspects of the diseases causing nephrocalcinosis have been elucidated, little is known about the cellular events in this calcification process. We previously reported a phenomenon involving the spontaneous formation of Ca2PO4 nodules in primary papillary renal cells from a patient with medullary nephrocalcinosis harboring a rare glial cell-derived neurotrophic factor (GDNF) gene variant. We also demonstrated that cultivating GDNF-silenced human kidney-2 (HK-2) cells in osteogenic conditions for 15 days triggered Ca2PO4 deposits. Given the reportedly close relationship between cell death and pathological calcification, aim of the present study was to investigate whether apoptosis is involved in the calcification of GDNF-silenced HK-2 cells under osteogenic conditions. Silenced and control cells were cultured in standard and osteogenic medium for 1, 5, and 15 days, and any Ca2PO4 deposition was identified by means of von Kossa staining and environmental SEM (ESEM) analyses. Based on the results of annexin V and propidium iodide (PI) analysis, and terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) assay, the silenced cells in the osteogenic medium showed a significant increase in the percentage of cells in the late phase of apoptosis and an increased Ca2PO4 deposition at 15 days. The results of quantitative real-time PCR (qRT-PCR) of BAX and BCL2, and in-cell Western analysis of caspases indicated that the cell death process was independent of caspase-3, -6, -7, and -9 activation, however. Using this model, we provide evidence of caspase-independent cell death triggering the calcification process in GDNF-silenced HK-2 cells.
Asunto(s)
Fosfatos de Calcio/metabolismo , Muerte Celular/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Nefrocalcinosis/genética , Apoptosis/genética , Caspasas/genética , Línea Celular , Células Epiteliales/metabolismo , Células Epiteliales/patología , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Nefrocalcinosis/metabolismo , Nefrocalcinosis/patologíaRESUMEN
BACKGROUND: Understanding how mesenchymal cells arise from epithelial cells could have a strong impact in unveiling mechanisms of epithelial cell plasticity underlying kidney regeneration and repair. In primary human tubular epithelial cells (HUTEC) under different TGF beta 1 concentrations we had observed epithelial-to-mesenchymal transition (EMT) but not epithelial-myofibroblast transdifferentiation. We hypothesized that the process triggered by TGFbeta 1 could be a dedifferentiation event. The purpose of this study is to comprehensively delineate genetic programs associated with TGF beta 1-driven EMT in our in vitro model using gene expression profile on large-scale oligonucleotide microarrays. RESULTS: In HUTEC under TGF beta 1 stimulus, 977 genes were found differentially expressed. Thirty genes were identified whose expression depended directly on TGF beta 1 concentration. By mapping the differentially expressed genes in the Human Interactome Map using Cytoscape software, we identified a single scale-free network consisting of 2630 interacting proteins and containing 449 differentially expressed proteins. We identified 27 hub proteins in the interactome with more than 29 edges incident on them and encoded by differentially expressed genes. The Gene Ontology analysis showed an excess of up-regulated proteins involved in biological processes, such as "morphogenesis", "cell fate determination" and "regulation of development", and the most up-regulated genes belonged to these categories. In addition, 267 genes were mapped to the KEGG pathways and 14 pathways with more than nine differentially expressed genes were identified. In our model, Smad signaling was not the TGF beta 1 action effector; instead, the engagement of RAS/MAPK signaling pathway seems mainly to regulate genes involved in the cell cycle and proliferation/apoptosis. CONCLUSION: Our present findings support the hypothesis that context-dependent EMT generated in our model by TGF beta 1 might be the outcome of a dedifferentiation. In fact: 1) the principal biological categories involved in the process concern morphogenesis and development; 2) the most up-regulated genes belong to these categories; and, finally, 3) some intracellular pathways are involved, whose engagement during kidney development and nephrogenesis is well known. These long-term effects of TGF beta 1 in HUTEC involve genes that are highly interconnected, thereby generating a scale-free network that we named the "TGF beta 1 interactome", whose hubs represent proteins that may have a crucial role for HUTEC in response to TGF beta 1.
Asunto(s)
Células Epiteliales/citología , Túbulos Renales/metabolismo , Mesodermo/citología , Factor de Crecimiento Transformador beta1/farmacología , Diferenciación Celular , Células Cultivadas , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Túbulos Renales/citología , Túbulos Renales/efectos de los fármacos , Mesodermo/metabolismo , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Albumin re-uptake is a receptor-mediated pathway located in renal proximal tubuli. There is increasing evidence of glomerular protein handling by podocytes, but little is known about the mechanism behind this process. In this study, we found that human podocytes in vitro are committed to internalizing albumin through a receptor-mediated mechanism even after exposure to low doses of albumin. We show that these cells express cubilin, megalin, ClC-5, amnionless and Dab2, which are partners in the tubular machinery. Exposing human podocytes to albumin overload prompted an increase in CUBILIN, AMNIONLESS and CLCN5 gene expression. Inhibiting cubilin led to a reduction in albumin uptake, highlighting its importance in this mechanism. We demonstrated that human podocytes are committed to performing endocytosis via a receptor-mediated mechanism even in the presence of low doses of albumin. We also disclosed that protein overload first acts on the expression of the cubilin-amnionless (CUBAM) complex in these cells, then involves the ClC-5 channel, providing the first evidence for a possible role of the CUBAM complex in albumin endocytosis in human podocytes.
Asunto(s)
Albúminas/metabolismo , Podocitos/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis , Transporte Biológico/fisiología , Células Cultivadas , Canales de Cloruro/metabolismo , Endocitosis/fisiología , Regulación de la Expresión Génica , Humanos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas de la Membrana , Complejos Multiproteicos/metabolismo , Podocitos/citología , Proteínas/metabolismo , Receptores de Albúmina/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Pentraxin 3 (PTX3) is an acute-phase protein involved in C1q clearance. The presence of anti-C1q and the absence of anti-PTX3 antibodies were associated with lupus glomerulonephritis (LGLN). Our aim was to assess soluble and kidney-expressed PTX3 and their relationships with anti-C1q and anti-PTX3 antibodies in LGLN. Serum PTX3, anti-C1q, anti-dsDNA, and anti-PTX3 antibodies were tested in 130 systemic lupus erythematosus (SLE) patients, 130 healthy and 127 disease controls. Twenty-nine renal biopsies from SLE patients were analyzed and PTX3 immunostaining was quantified by morphometric analysis. Parametric and nonparametric statistics were performed. PTX3 serum levels were lower in SLE versus controls, but they were correlated with proteinuria in LGLN patients (p = 0.001). LGLN patients had higher anti-C1q and lower anti-PTX3 antibody levels than those without (p < 0.0001). LGLN was more prevalent in anti-C1q(+)/anti-PTX3(-) than in anti-C1q(+)/anti-PTX3(+) patients (p < 0.001). No LGLN was observed in anti-C1q(-)/anti-PTX3(+) patients. PTX3 was expressed in glomeruli and renal interstitium. Renal PTX3 was correlated with proteinuria (p = 0.024) and interstitial fibrosis (p = 0.023). PTX3 staining and fibrosis were higher in anti-PTX3(-) than anti-PTX3(+) patients. In conclusion, PTX3 is expressed in glomeruli of LGLN patients, primarily in anti-PTX3(-) patients, where it is correlated with renal fibrosis. Anti-C1q/anti-PTX3 antibody profile seems to be useful in LGLN assessment.