RESUMEN
BACKGROUND: Increased bone loss has been associated with the development of vascular calcification in patients with chronic renal failure (CRF). In this study, the effect of impaired bone metabolism on aortic calcifications was investigated in uremic rats with or without ovariectomy. METHODS: CRF was induced by administration of a 0.75% adenine/2.5% protein diet for 4 weeks. In one group, osteoporosis was induced by ovariectomy (CRF-OVX), while the other group underwent a sham-operation instead (CRF). A third group consisted of ovariectomized rats with normal renal function (OVX). At regular time intervals throughout the study, bone status and aortic calcifications were evaluated by in vivo micro-CT. At sacrifice after 6 weeks of CRF, bone histomorphometry was performed and vascular calcification was assessed by bulk calcium analysis and Von Kossa staining. RESULTS: Renal function was significantly impaired in the CRF-OVX and CRF groups. Trabecular bone loss was seen in all groups. In the CRF-OVX and CRF groups, trabecular bone density was restored after adenine withdrawal, which coincided with cortical bone loss and the development of medial calcifications in the aorta. No significant differences with regard to the degree of aortic calcifications were seen between the two CRF groups. Neither cortical bone loss nor calcifications were seen in the OVX group. Cortical bone loss significantly correlated with the severity of vascular calcification in the CRF-OVX and CRF groups, but no associations with trabecular bone changes were found. CONCLUSIONS: Cortical rather than trabecular bone loss is associated with the process of calcification in rats with adenine- induced CRF.
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Calcinosis/patología , Fallo Renal Crónico/fisiopatología , Calcificación Vascular/patología , Adenina/farmacología , Animales , Aorta/patología , Peso Corporal , Huesos/patología , Progresión de la Enfermedad , Femenino , Osteoporosis/fisiopatología , Ovariectomía , Ratas , Ratas Wistar , Microtomografía por Rayos X/métodosRESUMEN
Pyrophosphate, a ubiquitous small-molecule inhibitor of mineralization abundantly present in the extracellular environment, binds to calcium and mineral surfaces to inhibit crystal growth. O'Neill and colleagues show in uremic rats that systemic administration of pyrophosphate prevents or reduces uremia-related vascular calcification, without overt negative consequences for bone and without calcium pyrophosphate deposition disease. These findings prompt further research into the potential of pyrophosphate as treatment for vascular calcification in chronic kidney disease patients.
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Calcinosis/prevención & control , Difosfatos/uso terapéutico , Uremia/complicaciones , Enfermedades Vasculares/prevención & control , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/fisiología , Animales , Difosfatos/metabolismo , Humanos , Osteopontina/fisiología , RatasRESUMEN
OBJECTIVE: To investigate cell biological changes in calcified aortas of rats that experienced chronic renal failure. METHODS AND RESULTS: Vascular smooth muscle cells have the potential to transdifferentiate to either chondrocytes or osteoblasts, depending on the molecular pathways that are stimulated. Uremia-related medial calcification was induced by feeding rats an adenine low-protein diet for 4 weeks. Aortic calcification was evaluated biochemically and histochemically and with in vivo micro-computed tomographic scanning. Immunohistochemistry and RT-PCR were applied to analyze the time-dependent aortic expression of molecules involved in the segregation between the chondrocyte versus osteoblast differentiation pathway. After 4 weeks, 85% of the uremic rats had developed distinct aortic medial calcification, which increased to severely calcified lesions during further follow-up. The calcification process was accompanied by a significant time-dependent increase in the expression of the chondrocyte-specific markers sex determining region Y-box 9 (sox9), collagen II, and aggrecan and a nonsignificant trend toward enhanced core binding factor alpha 1 (cbfa1), and collagen I. The expression of the osteoblast marker osterix and both lipoprotein receptor-related protein 6 and beta-catenin, molecules of the wingless-type MMTV integration site family member (Wnt)/beta-catenin pathway induced during osteoblast differentiation, was suppressed. CONCLUSIONS: In the aorta of uremic rats, medial smooth muscle cells acquire a chondrocyte rather than osteoblast phenotype during the calcification process.
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Enfermedades de la Aorta/patología , Calcinosis/patología , Transdiferenciación Celular , Condrocitos/patología , Fallo Renal Crónico/patología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Osteoblastos/patología , Uremia/patología , Adenina , Animales , Enfermedades de la Aorta/diagnóstico por imagen , Enfermedades de la Aorta/etiología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Biomarcadores/sangre , Biomarcadores/orina , Calcinosis/diagnóstico por imagen , Calcinosis/etiología , Calcinosis/genética , Calcinosis/metabolismo , Calcio/sangre , Calcio/orina , Transdiferenciación Celular/genética , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Inmunohistoquímica , Fallo Renal Crónico/diagnóstico por imagen , Fallo Renal Crónico/etiología , Fallo Renal Crónico/genética , Fallo Renal Crónico/metabolismo , Masculino , Músculo Liso Vascular/diagnóstico por imagen , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Osteoblastos/metabolismo , Fenotipo , Fósforo Dietético , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Uremia/diagnóstico por imagen , Uremia/etiología , Uremia/genética , Uremia/metabolismo , Microtomografía por Rayos XRESUMEN
Experts from all continents discussed the present and future of nephrology and transplantation medicine in emerging countries during a 3-day conference, supported by the World Health Organization, the International Society of Nephrology, the Transplantation Society-Global Alliance for Transplantation and the Ministry of Health of the Republic of Mali. This conference was held in Bamako, Mali on December 4-6, 2008, and focused on prevention and treatment of chronic kidney disease in emerging countries. Apart from delivering high-quality medical and scientific knowledge, the meeting was mainly a call to action for emerging countries to start chronic kidney disease prevention and screening programs, develop end-stage renal disease registries and start or further elaborate transplantation programs. International as well as regional collaborations need to be stimulated and strengthened in order to allow emerging countries to acquire the information, technology, experience and skills necessary to achieve these ambitious goals.
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Logro , Países en Desarrollo , Salud Global , Trasplante de Riñón/tendencias , Insuficiencia Renal Crónica/cirugía , Humanos , Trasplante de Riñón/etnología , Malí , Insuficiencia Renal Crónica/etnologíaRESUMEN
Vascular calcification or ectopic mineralization in blood vessels is an active, cell-regulated process, increasingly recognized as a general cardiovascular risk factor. Remarkably, ectopic artery mineralization is frequently accompanied by decreased bone mineral density or disturbed bone turnover. This contradictory association, observed mainly in osteoporosis and chronic kidney disease, is called the 'calcification paradox'. Here, we review recent advances in our understanding of the calcification paradox, including protein expression patterns governing both normal and ectopic mineralization, the conversion of vascular smooth muscle cells to bone-like cells, and the regulatory pathways involved in both bone and vessel mineralization. Further elucidation of the mechanisms underlying the calcification paradox is crucial in order to develop preventive and therapeutic strategies to deal with vascular calcification and reduce the associated cardiovascular risk.
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Arterias/fisiopatología , Calcificación Fisiológica , Osteoporosis/etiología , Enfermedades Vasculares/complicaciones , Animales , Regulación de la Expresión Génica , Humanos , Osteoporosis/metabolismo , Osteoporosis/fisiopatología , Transducción de Señal , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/fisiopatologíaRESUMEN
BACKGROUND: Hyperphosphataemia is a risk factor for arterial calcification contributing to the high cardiovascular mortality in patients with chronic kidney disease. Calcium-based phosphate binders can induce hypercalcaemia and are associated with progression of vascular calcification. Therefore, the effect of lanthanum carbonate, a non-calcium phosphate binder, on the development of vascular calcification was investigated in uraemic rats. METHODS: Chronic renal failure (CRF) was induced by feeding rats an adenine-enriched diet for 4 weeks. After 2 weeks, 1% or 2% lanthanum carbonate was added to the diet for 6 weeks. Calcification in the aorta, carotid and femoral arteries was evaluated histomorphometrically, biochemically and by ex vivo micro-CT. Chondro-/osteogenic conversion of vascular smooth muscle cells was also analysed in the rat aorta. RESULTS: Treatment with 1% lanthanum carbonate (1% La) did not reduce vascular calcification, but in the 2% lanthanum carbonate (2% La) group vascular calcium content and area% Von Kossa positivity were decreased compared with control CRF rats. The aortic calcified volume measured with ex vivo micro-CT was significantly reduced in rats treated with 2% La. Although calcification was inhibited by treatment with 2% La, the chondrocyte transcription factor sox-9 was abundantly expressed in the aorta. CONCLUSION: Treatment of CRF rats with 2% La reduces the development of vascular calcification by adequate phosphate binding resulting in a decreased supply of phosphate as a substrate for vascular calcification.
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Calcinosis/prevención & control , Fallo Renal Crónico/tratamiento farmacológico , Fallo Renal Crónico/metabolismo , Lantano/uso terapéutico , Fosfatos/metabolismo , Enfermedades Vasculares/prevención & control , Animales , Arterias/efectos de los fármacos , Arterias/metabolismo , Arterias/patología , Huesos/efectos de los fármacos , Huesos/metabolismo , Huesos/patología , Calcinosis/metabolismo , Calcinosis/patología , Condrogénesis/efectos de los fármacos , Hiperfosfatemia/tratamiento farmacológico , Hiperfosfatemia/metabolismo , Hiperfosfatemia/patología , Fallo Renal Crónico/patología , Lantano/administración & dosificación , Masculino , Osteogénesis/efectos de los fármacos , Ratas , Ratas Wistar , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/patologíaRESUMEN
Elevated serum phosphate levels as a consequence of chronic kidney disease (CKD) contribute to the increased cardiovascular risk observed in dialysis patients. Protein restriction and dialysis fail to adequately prevent hyperphosphatemia, and in general treatment with oral phosphate binding agents is necessary in patients with advanced CKD. Phosphate plays a pivotal role in the development of vascular calcification, one of the factors contributing to increased cardiovascular risk in CKD patients. Treatment of hyperphosphatemia with standard calcium-based phosphate binders and vitamin D compounds can induce hypercalcemic episodes, increase the Ca × PO(4) product and thus add to the risk of ectopic mineralization. In this review, recent clinical as well as experimental data on lanthanum carbonate, a novel, non-calcium, non-resin phosphate binding agent are summarized. Although lanthanum is a metal cation no aluminium-like toxicity is observed since the bioavailability of lanthanum is extremely low and its metabolism differs from that of aluminium. Clinical studies now document the absence of toxic effects of lanthanum for up to 6 years of follow-up. The effects of lanthanum on bone, vasculature and brain are discussed and put in perspective with lanthanum pharmacokinetics.
RESUMEN
OBJECTIVE: Chronic renal failure (CRF) is associated with a 10- to 20-fold increase in cardiovascular risk. Vascular calcification is a prominent feature of cardiovascular disease in patients with end-stage renal failure and contributes to the excess mortality in this population. In this study, we explored in vivo X-ray microtomography (micro-CT) as a tool to detect and follow-up vascular calcifications in the aorta of living rats with adenine-induced CRF. METHODS AND RESULTS: With in vivo micro-CT, calcification of the aorta in uremic rats was clearly discernible on transversal virtual cross-sections. Micro-CT findings correlated well with tissue calcium content and histology. Repetitive scans in animals with light, moderate, and severe vascular calcification showed good reproducibility with minimal interference of motion artifacts. Moreover, both calcified volume and area could be quantified with this method. CONCLUSIONS: In vivo micro-CT scanning is a sensitive method to detect vascular calcifications in CRF rats, allowing follow-up and quantification of the development, and potential reversal during treatment, of vascular calcifications in living animals.
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Enfermedades de la Aorta/diagnóstico por imagen , Enfermedades de la Aorta/etiología , Calcinosis/diagnóstico por imagen , Calcinosis/etiología , Fallo Renal Crónico/complicaciones , Tomografía Computarizada por Rayos X , Animales , Aorta Torácica/metabolismo , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Calcinosis/metabolismo , Calcinosis/patología , Calcio/metabolismo , Estudios de Factibilidad , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Técnicas In Vitro , Modelos Cardiovasculares , Ratas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Índice de Severidad de la EnfermedadRESUMEN
Accumulation of inorganic phosphate due to renal functional impairment contributes to the increased cardiovascular mortality observed in dialysis patients. Phosphate plays a causative role in the development of vascular calcification in renal failure; treatment with calcium-based phosphate binders and vitamin D can further increase the Ca x PO(4) product and add to the risk of ectopic mineralization. The new generation of calcium-free phosphate binders, sevelamer and lanthanum, can control hyperphosphatemia without adding to the patients calcium load. In this article, the metabolism of lanthanum carbonate and its effects in bone, liver and brain are discussed. Although lanthanum is a metal cation its effects are not comparable to those of aluminum. Indeed, in clinical studies no toxic effects of lanthanum have been reported after up to four years of follow-up. The bioavailability of lanthanum is extremely low. The effects observed in bone are due to phosphate depletion, with no signs of direct bone toxicity yet observed in rats or humans. The liver is the main route of excretion for lanthanum carbonate, which can be localized in the lysosomes of hepatocytes. No lanthanum could be detected in brain tissue.
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Lantano/metabolismo , Proteínas de Unión a Fosfato/efectos de los fármacos , Animales , Disponibilidad Biológica , Huesos/efectos de los fármacos , Huesos/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Calcinosis/tratamiento farmacológico , Calcinosis/metabolismo , Ensayos Clínicos como Asunto , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Fallo Renal Crónico/tratamiento farmacológico , Fallo Renal Crónico/metabolismo , Lantano/farmacocinética , Hígado/efectos de los fármacos , Hígado/metabolismo , Proteínas de Unión a Fosfato/sangre , Proteínas de Unión a Fosfato/farmacocinéticaRESUMEN
INTRODUCTION: Beside lung transplantation, cardiopulmonary bypass, isolated lung perfusion and sleeve resection result in serious pulmonary ischemia-reperfusion injury, clinically known as acute respiratory distress syndrome. Very little is known about cells infiltrating the lung during ischemia-reperfusion. Therefore, a model of warm ischemia-reperfusion injury was applied to differentiate cellular infiltrates and to quantify tissue damage. METHODS: Fifty rats were randomized into eight groups. Five groups underwent warm ischemia for 60 min followed by 30 min and 1-4 hours of warm reperfusion. An additional group was flushed with the use of isolated lung perfusion after 4 hours of reperfusion. One of two sham groups was also flushed. Neutrophils and oedema were investigated by using samples processed with hematoxylin/eosin stain at a magnification of x500. Immunohistochemistry with antibody ED-1 (magnification x250) and antibody 1F4 (magnification x400) was applied to visualize macrophages and T cells. TdT-mediated dUTP nick end labelling was used for detecting apoptosis. Statistical significance was accepted at P < 0.05. RESULTS: Neutrophils were increased after 30 min until 4 hours of reperfusion as well as after flushing. A doubling in number of macrophages and a fourfold increase in T cells were observed after 30 min until 1 and 2 hours of reperfusion, respectively. Apoptosis with significant oedema in the absence of necrosis was seen after 30 min to 4 hours of reperfusion. CONCLUSIONS: After warm ischemia-reperfusion a significant increase in infiltration of neutrophils, T cells and macrophages was observed. This study showed apoptosis with serious oedema in the absence of necrosis after all periods of reperfusion.
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Macrófagos Alveolares/fisiología , Neutrófilos/fisiología , Daño por Reperfusión/patología , Síndrome de Dificultad Respiratoria/fisiopatología , Animales , Apoptosis/fisiología , Edema/fisiopatología , Masculino , Ratas , Ratas Wistar , Daño por Reperfusión/inmunología , Síndrome de Dificultad Respiratoria/inmunologíaRESUMEN
Inflammation has been established to contribute substantially to the pathogenesis of ischemia/reperfusion (I/R) with a central role for particular cells, adhesion molecules, and cytokines. Until recently, most of the research trying to unravel the pathogenesis of I/R injury has been focused on the role of neutrophils. However, recent studies have brought evidence that T cells and macrophages are also important leukocyte mediators of renal and extrarenal (liver) I/R injury. In vivo depletion of CD4+ cells but not CD8+ cells in wild-type mice was protective in I/R of the kidney. A marked preservation of liver function was also found after I/R in T-cell deficient athymic mice. Blocking the b130/CD28 costimulatory pathway by CTLA-4 Ig (recombinant fusion protein) ameliorated renal dysfunction and decreased mononuclear cell infiltration in I/R of the kidney. b130-1 expression was found limited to the membrane of the endothelial cells of the ascending vasa recta, resulting in trapping of CD28-expressing CD4 T cells. This trapping of leukocytes results in the upstream congestion in the ascending arterial vasa recta, generating the since more than 150 years described medullary vascular congestion of the kidney soon after ischemic injury. It seems worthwhile to study a combination therapy using anti-inflammatory/anti-adhesion molecules in the early phase of I/R.
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Lesión Renal Aguda/inmunología , Daño por Reperfusión/inmunología , Linfocitos T/inmunología , Lesión Renal Aguda/patología , Lesión Renal Aguda/fisiopatología , Animales , Humanos , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatologíaRESUMEN
BACKGROUND: After ischemia/reperfusion (I/R), as well as after toxic insults, there is significant infiltration of leukocytes in the kidney. It is well known that antibodies against adhesion molecules [e.g., intercellular adhesion molecule-1 (ICAM-1)] protect the kidney against acute ischemic injury. In contrast, same antibody treatment did not protect the rat kidney against toxic acute renal failure (ARF) induced by HgCl2. Protection obtained by anti-adhesion treatment in I/R injury is an early phenomenon, since delaying the administration of anti-ICAM-1 for 8 hours did not protect the kidney anymore. The aim of this study was to compare the early ICAM-1 expression and leukocyte accumulation in different zones of ischemic and toxic injury. METHODS: Male Lewis rats were injected with HgCl2 (2 mg/kg, subcutaneously) or uninephrectomized Lewis rats were submitted to 30 degrees C warm ischemia (I/R injury). Rats were sacrificed at 2, 6, 12 and 24 hours. ICAM-1 (1A29) expression in kidney was evaluated morphometrically. Different subsets of leukocytes were stained by immunohistochemistry and counted in cortex, the outer stripe of the outer medulla (OSOM) and the level of the inner stripe of the outer medulla (ISOM). RESULTS: Although the functional and morphologic damage was comparable between the I/R and toxic ARF group, different ICAM-1 expression could be observed early after injury. ICAM-1 expression in the ISOM started already 2 hours after the onset of I/R injury, and was increased after 12 hours in the cortex and after 24 hours in the OSOM. In contrast, during the first 24 hours after injury, ICAM-1 expression in HgCl2-injured kidneys was not different from noninjured kidneys in the ISOM and the cortex, whereas in the OSOM, ICAM-1 expression increased. The number of polymononuclear cells (PMNs) was low in noninjured kidneys and did not increase in time after both I/R injury and after HgCl2-induced ARF. In the ISOM, significant monocyte and T-cell accumulation was observed early after I/R but not after HgCl2. There was no significant T-cell accumulation in the cortex or in the OSOM. CONCLUSION: After HgCl2, almost no leukocyte accumulation and up-regulation of ICAM-1 was observed the first 12 hours after injury. In contrast, very early after I/R injury, increased expression of ICAM-1 goes along with monocyte and T-cell accumulation in the ISOM, endorsing this particular zone as critical in renal I/R injury. These observations contribute to the understanding why anti-ICAM-1 treatment in acute I/R injury is successful, but fails in acute toxic injury induced by HgCl2.
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Lesión Renal Aguda/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Isquemia/metabolismo , Leucocitos/citología , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/patología , Animales , Adhesión Celular , Isquemia/patología , Corteza Renal/metabolismo , Corteza Renal/patología , Médula Renal/metabolismo , Médula Renal/patología , Macrófagos/citología , Masculino , Cloruro de Mercurio , Monocitos/citología , Neutrófilos/citología , Ratas , Ratas Endogámicas Lew , Linfocitos T/citología , Regulación hacia ArribaRESUMEN
BACKGROUND: Osteopontin (OPN) is a phosphoprotein that is up-regulated in several experimental models of renal disease, including ischemia/reperfusion injury. OPN has been described as a macrophage chemoattractant, may serve as a survival factor for tubular cells, and is implicated in the development of tubulointerstitial fibrosis. However, the precise role of this protein in renal pathophysiology remains unclear. METHODS: OPN knockout and wild-type mice were subjected to 30 minutes of warm renal ischemia combined with a contralateral nephrectomy, and sacrificed at six different time points, ranging from 12 hours to seven days after reperfusion. Besides functional and morphological parameters of postischemic acute renal failure (ARF), macrophage infiltration, apoptosis and expression of collagen types I and IV were investigated. RESULTS: Postischemic ARF in OPN knockouts and wild-types showed a similar course and severity, without significant differences in either functional or morphological disease parameters. However, macrophage infiltration was significantly diminished in OPN knockouts after five and seven days, in cortex as well as in the outer stripe of the outer medulla (OSOM). Furthermore, OPN knockout mice showed significantly enhanced apoptosis in the injury phase and significantly less collagen I and IV expression in the regeneration phase of postischemic ARF. CONCLUSIONS: There was no influence of OPN protein on the severity or course of functional impairment or morphological injury in the first seven days after an ischemic insult to the kidney. However, our results demonstrate that OPN favors macrophage recruitment to the postischemic kidney, inhibits apoptosis, and stimulates the development of renal fibrosis after an acute ischemic insult.
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Isquemia/metabolismo , Isquemia/patología , Riñón/patología , Macrófagos/patología , Circulación Renal , Sialoglicoproteínas/deficiencia , Animales , Apoptosis , Temperatura Corporal , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Fibrosis , Inmunohistoquímica/métodos , Isquemia/fisiopatología , Riñón/fisiopatología , Ratones , Ratones Noqueados , Osteopontina , Regeneración , Coloración y EtiquetadoRESUMEN
Nephrolithiasis requires formation of crystals followed by their retention and accumulation in the kidney. Crystal retention can be caused by the association of crystals with the epithelial cells lining the renal tubules. The present study investigated the interaction between calcium oxalate monohydrate (COM) crystals and primary cultures of human proximal (PTC) and distal tubular/collecting duct cells (DTC). Both PTC and DTC were susceptible to crystal binding during the first days post-seeding (4.9 +/- 0.8 micro g COM/cm2), but DTC lost this affinity when the cultures developed into confluent monolayers with functional tight junctions (0.05 +/- 0.02 micro g COM/cm2). Confocal microscopy demonstrated the expression of the transmembrane receptor protein CD44 and its ligands osteopontin (OPN) and hyaluronic acid (HA) at the apical membrane of proliferating tubular cells; at confluence, CD44 was expressed at the basolateral membrane and OPN and HA were no longer detectable. In addition, a particle exclusion technique revealed that proliferating cells were surrounded by HA-rich pericellular matrices or "cell coats" extending several microns from the cell surface. Disintegration of these coats with hyaluronidase significantly decreased the cell surface affinity for crystals. Furthermore, CD44, OPN, and HA were also expressed in vivo at the luminal side of tubular cells in damaged kidneys. These results suggest (1) that the intact distal tubular epithelium of the human kidney does not bind crystals, and (2) that crystal retention in the human kidney may depend on the expression of CD44-, OPN-, and-HA rich cell coats by damaged distal tubular epithelium.
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Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Cálculos Renales/fisiopatología , Sialoglicoproteínas/metabolismo , Oxalato de Calcio/química , Oxalato de Calcio/metabolismo , Adhesión Celular , Células Cultivadas , Cristalización , Matriz Extracelular/metabolismo , Humanos , Riñón/patología , Riñón/fisiopatología , Cálculos Renales/patología , Túbulos Renales/metabolismo , Ligandos , Osteopontina , Distribución TisularRESUMEN
In normal human and rat kidneys, osteopontin (OPN) is present at the apical surface of cells in the distal nephron. After ischemic or toxic renal damage in rats, OPN is upregulated in distal tubular cells (DTC) and expressed de novo in perinuclear vesicles in proximal tubular cells (PTC). In the first phase of this study, OPN localization in ischemic human biopsies was compared with that in ischemic rat kidneys. In the second phase, cultures of PTC and DTC were used to investigate human renal OPN synthesis, secretion, and localization. OPN localization in human biopsies after renal ischemia was comparable to that in ischemic rat kidneys. Microscopic and flow cytometric detection of immunofluorescent OPN staining in tubular cell cultures demonstrated strong plasma membrane localization in DTC, whereas mainly perinuclear intracellular expression was observed in PTC. Northern blotting and reverse transcription-PCR demonstrated production of a single OPN mRNA in PTC and DTC. Detection of OPN by Western blotting and enzyme-linked immunosorbent assay demonstrated that PTC and DTC synthesized and secreted the same three molecular mass OPN forms, in comparable amounts. Finally, confocal microscopy demonstrated different staining patterns for endocytotic/lysosomal vesicles and perinuclear OPN; however, perinuclear OPN exhibited colocalization with the Golgi apparatus. In conclusion, human renal OPN localization in cell cultures demonstrated differences between PTC and DTC comparable to those observed after renal ischemia in vivo. Therefore, these cell cultures represented an excellent model for the study of human OPN synthesis, secretion, and localization in PTC versus DTC. It is reported for the first time that intracellular OPN is located in the Golgi apparatus of both PTC and DTC and that PTC and DTC are able to produce and secrete the same OPN isoforms, in comparable amounts.