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1.
Artículo en Inglés | MEDLINE | ID: mdl-32061840

RESUMEN

Short-chain fatty acids (SCFAs), including acetate, butyrate, and propionate, are produced when colonic bacteria in the human gastrointestinal tract ferment undigested fibers. Free fatty acid receptor 2 (FFA2) and FFA3 are G-protein-coupled receptors recently identified as SCFA receptors that may modulate inflammation. We previously showed through in vitro experiments that SCFAs activate FFA2 and FFA3, thereby mitigating inflammation in human renal cortical epithelial cells. This study used a murine model of adenine-induced renal failure to investigate whether or not SCFAs can prevent the progression of renal damage. We also examined whether or not these FFA2 and FFA3 proteins have some roles in this protective mechanism in vivo. Immunohistochemical analyses of mouse kidneys showed that FFA2 and FFA3 proteins were expressed mainly in the distal renal tubules and collecting tubules. First, we observed that the administration of propionate mitigated the renal dysfunction and pathological deterioration caused by adenine. Consistent with this, the expression of inflammatory cytokines and fibrosis-related genes was reduced. Furthermore, the mitigation of adenine-induced renal damage by the administration of propionate was significantly attenuated in FFA2-/- and FFA3-/- mice. Therefore, the administration of propionate significantly protects against adenine-induced renal failure, at least in part, via the FFA2 and FFA3 pathways. Our data suggest that FFA2 and FFA3 are potential new therapeutic targets for preventing or delaying the progression of chronic kidney disease.


Asunto(s)
Propionatos/administración & dosificación , Receptores Acoplados a Proteínas G/metabolismo , Insuficiencia Renal Crónica/prevención & control , Adenina/toxicidad , Animales , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/inmunología , Túbulos Renales Colectores/patología , Túbulos Renales Distales/efectos de los fármacos , Túbulos Renales Distales/inmunología , Túbulos Renales Distales/patología , Masculino , Ratones , Ratones Noqueados , Receptores Acoplados a Proteínas G/genética , Insuficiencia Renal Crónica/inducido químicamente , Insuficiencia Renal Crónica/inmunología , Insuficiencia Renal Crónica/patología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología
2.
Am J Physiol Renal Physiol ; 318(2): F468-F474, 2020 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-31841391

RESUMEN

Acute pyelonephritis is frequently associated with metabolic acidosis. We previously reported that metabolic acidosis stimulates expression of hypoxia-inducible factor (HIF)-1α-induced target genes such as stromal derived factor-1 and cathelicidin, an antimicrobial peptide. Since the collecting duct (CD) plays a pivotal role in regulating acid-base homeostasis and is the first nephron segment encountered by an ascending microbial infection, we examined the contribution of HIF-1α to innate immune responses elicited by acid loading of an M-1 immortalized mouse CD cell line. Acid loading of confluent M-1 cells was achieved by culture in pH 6.8 medium supplemented with 5-(N-ethyl-N-isopropyl)-amiloride to block Na+/H+ exchange activity for 24 h. Acid loading induced antimicrobial peptide [cathelicidin and ß-defensin (Defb2 and Defb26)] mRNA expression and M-1 cell resistance to uropathogenic Escherichia coli infection to an extent similar to that obtained by inhibition of HIF prolyl hydroxylases, which promote HIF-1α protein degradation. The effect of acid loading on M-1 cell resistance to uropathogenic E. coli infection was reduced by inhibition of HIF-1α (PX-478), and, in combination with prolyl hydroxylase inhibitors, acidosis did not confer additional resistance. Thus, metabolic stress of acidosis triggers HIF-1α-dependent innate immune responses in CD (M-1) cells. Whether pharmacological stabilization of HIF prevents or ameliorates pyelonephritis in vivo warrants further investigation.


Asunto(s)
Acidosis/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Infecciones por Escherichia coli/prevención & control , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Túbulos Renales Colectores/metabolismo , Infecciones Urinarias/prevención & control , Escherichia coli Uropatógena/patogenicidad , Acidosis/inmunología , Animales , Péptidos Catiónicos Antimicrobianos/genética , Línea Celular , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Interacciones Huésped-Patógeno , Inmunidad Innata , Túbulos Renales Colectores/inmunología , Túbulos Renales Colectores/microbiología , Ratones , Prolil Hidroxilasas/metabolismo , Estabilidad Proteica , Transducción de Señal , Regulación hacia Arriba , Infecciones Urinarias/inmunología , Infecciones Urinarias/metabolismo , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/inmunología , beta-Defensinas/metabolismo , Catelicidinas
3.
Sci Rep ; 9(1): 545, 2019 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-30679625

RESUMEN

The renal collecting duct consists of intercalated cells (ICs) and principal cells (PCs). We have previously demonstrated that collecting ducts have a role in the innate immune defense of the kidney. Transcriptomics is an important tool used to enhance systems-level understanding of cell biology. However, transcriptomics performed on whole kidneys provides limited insight of collecting duct cell gene expression, because these cells comprise a small fraction of total kidney cells. Recently we generated reporter mouse models to enrich collecting duct specific PC and ICs and reported targeted gene expression of anti-microbial peptide genes. Here we report transcriptomics on enriched ICs and PCs and performed a pilot study sequencing four single ICs. We identified 3,645 genes with increased relative expression in ICs compared to non-ICs. In comparison to non-PCs, 2,088 genes had higher relative expression in PCs. IC associated genes included the innate interleukin 1 receptor, type 1 and the antimicrobial peptide(AMP) adrenomedullin. The top predicted canonical pathway for enriched ICs was lipopolysaccharide/Interleukin 1 mediated inhibition of Retinoid X Receptor alpha function and decreased Retinoid X Receptor expression was confirmed to occur 1-hour post experimental murine UTI in ICs but not in non-ICs.


Asunto(s)
Células Epiteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/inmunología , Lipopolisacáridos/metabolismo , Receptor alfa X Retinoide/antagonistas & inhibidores , Receptor alfa X Retinoide/metabolismo , Animales , Acuaporina 2/genética , Acuaporina 2/metabolismo , Femenino , Inmunidad Innata/genética , Interleucina-1/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proyectos Piloto , Transducción de Señal/genética , Transcriptoma/inmunología , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo
4.
Am J Physiol Renal Physiol ; 315(4): F812-F823, 2018 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-28468965

RESUMEN

The urinary tract is usually culture negative despite its close proximity to microbial flora. The precise mechanism by which the kidneys and urinary tract defends against infection is not well understood. The initial kidney cells to encounter ascending pathogens are the collecting tubule cells that consist of principal cells (PCs) that express aquaporin 2 (AQP2) and intercalated cells (ICs) that express vacuolar H+-ATPase (V-ATPase, B1 subunit). We have previously shown that ICs are involved with the human renal innate immune defense. Here we generated two reporter mice, VATPase B1-cre+tdT+ mice to fluorescently label ICs and AQP2-cre+tdT+ mice to fluorescently label PCs, and then performed flow sorting to enrich PCs and ICs for analysis. Isolated ICs and PCs along with proximal tubular cells were used to measure antimicrobial peptide (AMP) mRNA expression. ICs and PCs were significantly enriched for AMPs. Isolated ICs responded to uropathogenic Escherichia coli (UPEC) challenge in vitro and had higher RNase4 gene expression than control while both ICs and PCs responded to UPEC challenge in vivo by upregulating Defb1 mRNA expression. To our knowledge, this is the first report of isolating murine collecting tubule cells and performing targeted analysis for multiple classes of AMPs.


Asunto(s)
Acuaporina 2/inmunología , Células Epiteliales/metabolismo , Túbulos Renales Colectores/inmunología , Reacción en Cadena de la Polimerasa , Animales , Acuaporina 2/genética , Inmunidad Innata/inmunología , Riñón/inmunología , Riñón/metabolismo , Ratones Transgénicos , Reacción en Cadena de la Polimerasa/métodos , Regulación hacia Arriba/inmunología , ATPasas de Translocación de Protón Vacuolares/inmunología , ATPasas de Translocación de Protón Vacuolares/metabolismo
5.
Mol Cell Biol ; 37(22)2017 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-28807932

RESUMEN

Acute kidney injury (AKI) is a leading cause of chronic kidney disease. Proximal tubules are considered to be the primary origin of pathogenic inflammatory cytokines in AKI. However, it remains unclear whether other cell types, including collecting duct (CD) cells, participate in inflammatory processes. The transcription factor GATA2 is specifically expressed in CD cells and maintains their cellular identity. To explore the pathophysiological function of GATA2 in AKI, we generated renal tubular cell-specific Gata2 deletion (G2CKO) mice and examined their susceptibility to ischemia reperfusion injury (IRI). Notably, G2CKO mice exhibited less severe kidney damage, with reduced granulomacrophagic infiltration upon IRI. Transcriptome analysis revealed that a series of inflammatory cytokine genes were downregulated in GATA2-deficient CD cells, suggesting that GATA2 induces inflammatory cytokine expression in diseased kidney CD cells. Through high-throughput chemical library screening, we identified a potent GATA inhibitor. The chemical reduces cytokine production in CD cells and protects the mouse kidney from IRI. These results revealed a novel pathological mechanism of renal IRI, namely, that CD cells produce inflammatory cytokines and promote IRI progression. In injured kidney CD cells, GATA2 exerts a proinflammatory function by upregulating inflammatory cytokine gene expression. GATA2 can therefore be considered a therapeutic target for AKI.


Asunto(s)
Lesión Renal Aguda/genética , Citocinas/genética , Factor de Transcripción GATA2/genética , Túbulos Renales Colectores/inmunología , Lesión Renal Aguda/inmunología , Animales , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Ensayos Analíticos de Alto Rendimiento , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión
6.
J Am Soc Nephrol ; 27(10): 3220-3228, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26984885

RESUMEN

Tubulointerstitial nephritis is a common cause of kidney failure and may have diverse etiologies. This form of nephritis is sometimes associated with autoimmune disease, but the role of autoimmune mechanisms in disease development is not well understood. Here, we present the cases of three patients with autoimmune polyendocrine syndrome type 1 who developed tubulointerstitial nephritis and ESRD in association with autoantibodies against kidney collecting duct cells. One of the patients developed autoantibodies targeting the collecting duct-specific water channel aquaporin 2, whereas autoantibodies of the two other patients reacted against the HOXB7 or NFAT5 transcription factors, which regulate the aquaporin 2 promoter. Our findings suggest that tubulointerstitial nephritis developed in these patients as a result of an autoimmune insult on the kidney collecting duct cells.


Asunto(s)
Acuaporinas/inmunología , Autoanticuerpos/inmunología , Túbulos Renales Colectores/inmunología , Nefritis Intersticial/inmunología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven
7.
Inflammation ; 39(1): 87-95, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26260258

RESUMEN

Guggulsterone (GS) is a phytosterol that has been used to treat inflammatory diseases such as colitis, obesity, and thrombosis. Although many previous studies have examined activities of GS, the effect of GS on lipopolysaccharide (LPS)-induced inflammatory responses in mouse inner medullary collecting duct-3 (mIMCD-3) cells have not been examined. Therefore, here, we investigated the anti-inflammatory action of GS on mIMCD-3 cells exposed to LPS. LPS treatment on mIMCD-3 cells produced pro-inflammatory molecules such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) significantly; however, GS treatment significantly inhibited the production of pro-inflammatory molecules. In addition, GS inhibited the degradation of Iκ-Bα and translocation of NF-κB on mIMCD-3 cells. These results suggest that GS could inhibit inflammatory responses in collecting duct cells which could contribute to kidney injury during systemic infection.


Asunto(s)
Lesión Renal Aguda/tratamiento farmacológico , Antiinflamatorios/farmacología , Túbulos Renales Colectores/inmunología , Pregnenodionas/farmacología , Receptor Toll-Like 4/inmunología , Animales , Línea Celular , Ciclooxigenasa 2/biosíntesis , Femenino , Inflamación/tratamiento farmacológico , Interleucina-6/biosíntesis , Túbulos Renales Colectores/citología , Lipopolisacáridos , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos C57BL , Inhibidor NF-kappaB alfa/metabolismo , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/biosíntesis
8.
Clin J Am Soc Nephrol ; 10(2): 305-24, 2015 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-25632105

RESUMEN

Intercalated cells are kidney tubule epithelial cells with important roles in the regulation of acid-base homeostasis. However, in recent years the understanding of the function of the intercalated cell has become greatly enhanced and has shaped a new model for how the distal segments of the kidney tubule integrate salt and water reabsorption, potassium homeostasis, and acid-base status. These cells appear in the late distal convoluted tubule or in the connecting segment, depending on the species. They are most abundant in the collecting duct, where they can be detected all the way from the cortex to the initial part of the inner medulla. Intercalated cells are interspersed among the more numerous segment-specific principal cells. There are three types of intercalated cells, each having distinct structures and expressing different ensembles of transport proteins that translate into very different functions in the processing of the urine. This review includes recent findings on how intercalated cells regulate their intracellular milieu and contribute to acid-base regulation and sodium, chloride, and potassium homeostasis, thus highlighting their potential role as targets for the treatment of hypertension. Their novel regulation by paracrine signals in the collecting duct is also discussed. Finally, this article addresses their role as part of the innate immune system of the kidney tubule.


Asunto(s)
Equilibrio Ácido-Base , Células Epiteliales/fisiología , Túbulos Renales Colectores/fisiología , Acidosis Tubular Renal/metabolismo , Acidosis Tubular Renal/fisiopatología , Animales , Diferenciación Celular , Linaje de la Célula , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Humanos , Inmunidad Innata , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/inmunología , Túbulos Renales Colectores/metabolismo , Fenotipo , Sistema Renina-Angiotensina
9.
Cell Microbiol ; 16(10): 1503-17, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24779433

RESUMEN

Uropathogenic Escherichia coli (UPEC) colonizing kidneys is the main cause of acute pyelonephritis. TLR5 that senses flagellin was shown to be highly expressed in the bladder and to participate in host defence against flagellated UPEC, although its role in kidneys still remains elusive. Here we show that TLR5 is expressed in renal medullary collecting duct (MCD) cells, which represent a preferential site of UPEC adhesion. Flagellin, like lipopolysaccharide, stimulated the production of the chemoattractant chemokines CXCL1 and CXCL2, and subsequent migration capacity of neutrophils in cultured wild-type (WT) and Tlr4(-/-) MCDs, but not in Tlr5(-/-) MCDs. UPEC can translocate across intact MCD layers without altering tight junctions. Strikingly, the invasion capacity and transcellular translocation of the UPEC strain HT7 were significantly lower in Tlr5(-/-) than in WT MCDs. The non-motile HT7ΔfliC mutant lacking flagellin also exhibited much lower translocation capacities than the HT7 isolates. Finally, Tlr5(-/-) kidneys exhibited less infiltrating neutrophils than WT kidneys one day after the transurethral inoculation of HT7, and greater delayed renal bacterial loads in the day 4 post-infected Tlr5(-/-) kidneys. Overall, these findings indicate that the epithelial TLR5 participates to renal antibacterial defence, but paradoxically favours the translocation of UPEC across intact MCD cell layers.


Asunto(s)
Infecciones por Escherichia coli/inmunología , Flagelina/inmunología , Túbulos Renales Colectores/inmunología , Receptor Toll-Like 5/inmunología , Escherichia coli Uropatógena/patogenicidad , Animales , Adhesión Bacteriana/fisiología , Carga Bacteriana/inmunología , Quimiocina CXCL1/biosíntesis , Quimiocina CXCL2/biosíntesis , Infecciones por Escherichia coli/microbiología , Femenino , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila/genética , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Pielonefritis/inmunología , Pielonefritis/microbiología , Transducción de Señal , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 5/genética , Vejiga Urinaria/inmunología , Vejiga Urinaria/metabolismo , Infecciones Urinarias/inmunología , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/inmunología
10.
Cell Microbiol ; 13(8): 1107-13, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21615666

RESUMEN

Urinary tract infections (UTIs), which are mainly due to uropathogenic Escherichia coli (UPEC), occur via the retrograde ascent of the bacteria along the urinary tract system. The adhesion and invasion mechanisms of UPEC have been extensively studied in bladder epithelial cells, but less is known about the role of renal tubule epithelial cells (RTEC) in renal antibacterial defences. This review considers recent advances in the understanding of the role of RTECs in inducing an innate immune response mediated by Toll-like receptors (TLRs) in experimental UTI. Collecting duct cells are a preferential site of adhesion of UPEC colonizing the kidneys. Epithelial TLR4 activation induces an inflammatory response and the recruitment of lipid rafts to the plasma membrane, both of which facilitate the transcytosis of non-cytolytic UPEC strains across intact collecting duct cell layers to invade the renal interstitium. Arginine vasopressin, which regulates water absorption in the collecting duct, also acts as a potent modulator of the TLR4-mediated intrarenal innate response caused by UPEC. The role of epithelial TLR5 in renal host defences is also discussed. These findings highlight the role of RTECs in triggering the innate immune response in the context of ascending UTIs.


Asunto(s)
Células Epiteliales/inmunología , Infecciones por Escherichia coli/inmunología , Túbulos Renales Colectores/inmunología , Infecciones Urinarias/inmunología , Escherichia coli Uropatógena/inmunología , Arginina Vasopresina/metabolismo , Adhesión Bacteriana , Humanos , Inmunidad Innata , Modelos Biológicos , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 5/metabolismo , Transcitosis , Escherichia coli Uropatógena/patogenicidad
11.
Biomarkers ; 15(5): 424-35, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20491521

RESUMEN

Currently there are no biomarkers for detecting collecting duct damage in man. Antibodies to several collecting duct-specific antigens exist but sandwich assays have been difficult to establish due to the need for two different antibodies to the same protein. We hypothesized that a collecting duct-specific lectin could be used in combination with a collecting duct-specific antibody to negate the need for two different antibodies. The collecting duct specificity of selected antibodies (NiCa II 13C2, Pap XI 3C7, HuPaP VII 2B11 and aquaporin 2), was verified by immunohistochemistry. Aquaporin 2 and Pap XI 3C7 were used successfully in setting up assays with the lectin Dolichos biflorus, using the Meso Scale Discovery (MSD) platform. Antigen expression was highest in the papillae of rat and human kidney (corresponding to the greatest density of collecting ducts) and was also present in normal urine. We propose that further qualification and validation would lead to an assay for detecting collecting duct damage in man.


Asunto(s)
Anticuerpos/análisis , Biomarcadores/análisis , Inmunoensayo/métodos , Túbulos Renales Colectores/inmunología , Lectinas de Plantas/inmunología , Animales , Antígenos/orina , Acuaporina 2/inmunología , Etilaminas , Humanos , Inmunohistoquímica , Riñón/inmunología , Riñón/metabolismo , Necrosis Papilar Renal/inducido químicamente , Necrosis Papilar Renal/inmunología , Necrosis Papilar Renal/orina , Masculino , Ratas , Ratas Wistar
12.
J Biomed Biotechnol ; 2010: 696034, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20037736

RESUMEN

Epithelial-mesenchymal transition (EMT) has emerged in recent years as an important process in the development of organ fibrosis in many human diseases. Our previous experience in a nonhuman primate model of obstructive nephropathy suggested that EMT of collecting duct epithelium contributes to the development of interstitial fibrosis. In this study we demonstrate for the first time in humans that obstructed fetal collecting duct epithelium undergoes transition to mesenchymal phenotype, characterized by decreased expression of epithelial markers, de novo expression of mesenchymal markers with subsequent loss of cell-cell interaction, disruption of the basement membrane, and increased deposition of extracellular matrix into the expanded interstitium of the obstructed kidney. The results of this study therefore support the previous findings from animal studies and suggest that EMT of the collecting duct epithelium might contribute to the development of interstitial fibrosis in human fetal obstructive nephropathy.


Asunto(s)
Epitelio/inmunología , Epitelio/patología , Túbulos Renales Colectores/inmunología , Túbulos Renales Colectores/patología , Obstrucción del Cuello de la Vejiga Urinaria/congénito , Obstrucción del Cuello de la Vejiga Urinaria/patología , Diferenciación Celular , Humanos , Fenotipo , Obstrucción del Cuello de la Vejiga Urinaria/inmunología
13.
Infect Immun ; 77(3): 959-69, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19124603

RESUMEN

Hemolytic-uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli infection is a leading cause of pediatric acute renal failure. Bacterial toxins produced in the gut enter the circulation and cause a systemic toxemia and targeted cell damage. It had been previously shown that injection of Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) caused signs and symptoms of HUS in mice, but the mechanism leading to renal failure remained uncharacterized. The current study elucidated that murine cells of the glomerular filtration barrier were unresponsive to Stx2 because they lacked the receptor glycosphingolipid globotriaosylceramide (Gb(3)) in vitro and in vivo. In contrast to the analogous human cells, Stx2 did not alter inflammatory kinase activity, cytokine release, or cell viability of the murine glomerular cells. However, murine renal cortical and medullary tubular cells expressed Gb(3) and responded to Stx2 by undergoing apoptosis. Stx2-induced loss of functioning collecting ducts in vivo caused production of increased dilute urine, resulted in dehydration, and contributed to renal failure. Stx2-mediated renal dysfunction was ameliorated by administration of the nonselective caspase inhibitor Q-VD-OPH in vivo. Stx2 therefore targets the murine collecting duct, and this Stx2-induced injury can be blocked by inhibitors of apoptosis in vivo.


Asunto(s)
Apoptosis/fisiología , Epitelio/patología , Túbulos Renales Colectores/patología , Toxina Shiga II/metabolismo , Animales , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Western Blotting , Células Cultivadas , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Epitelio/inmunología , Epitelio/metabolismo , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/patología , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Túbulos Renales Colectores/inmunología , Túbulos Renales Colectores/metabolismo , Lipopolisacáridos/inmunología , Ratones , Toxina Shiga II/inmunología , Venas Umbilicales
14.
Cell Physiol Biochem ; 21(4): 269-78, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18441515

RESUMEN

In this study, we demonstrate that a putative membrane unknown solute transporter 1 of the rat kidney (UST1r; Slc22a9) is a multispecific transporter of organic anions (OAs). When expressed in Xenopus oocytes, UST1r mediated uptake of ochratoxin A (OTA; K(m) = 1.0 microM) and sulfate conjugates of steroids, such as estrone-3-sulfate (ES; K(m) = 3.1 microM) and dehydroepiandrosterone sulfate (DHEAS; K(m) = 2.1 microM) in a sodium-independent manner. We herein propose that UST1r be renamed OA transporter 8 (rOat8). rOat8 interacted with chemically heterogenous anionic compounds, such as nonsteroidal anti-inflammatory drugs, diuretics, probenecid, taurocholate, and methotrexate, but not with the organic cation tetraethylammonium. The rOat8-mediated ES transport was: a) cis-inhibited by 4-methylumbelliferyl sulfate and beta-estradiol sulfate, but not by glucuronide conjugates of these compounds, b) cis-inhibited by four- and five- carbon (C4/C5) dicarboxylates (succinate and glutarate (GA)), and c) trans-stimulated by GA, whereas the efflux of GA was significantly trans-stimulated by ES. By RT-PCR, rOat8 mRNA was expressed in proximal convoluted tubules and cortical and outer medullary collecting ducts, whereas in immunochemical studies, Oat8 was identified as the ñ58 kDa protein that in the collecting duct colocalized with the V-ATPase in plasma membranes and intracellular vesicles in various subtypes of intercalated cells. Molecular identification of Oat8 in these cells indicates a possible novel role of OAT family in the renal secretion/reabsorption of OA and acids and bases via affecting the V-ATPase-dependent functions.


Asunto(s)
Túbulos Renales Colectores/inmunología , Túbulos Renales Colectores/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Transportadores de Anión Orgánico/metabolismo , Animales , Transporte Biológico , Femenino , Inmunohistoquímica , Masculino , Oocitos , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico Sodio-Independiente/genética , Ratas , Ratas Wistar , Xenopus laevis
15.
Mol Biol Cell ; 19(4): 1587-93, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18256279

RESUMEN

In mammals the transfer of passive immunity from mother to young is mediated by the MHC-related receptor FcRn, which transports maternal IgG across epithelial cell barriers. In birds, maternal IgY in egg yolk is transferred across the yolk sac to passively immunize chicks during gestation and early independent life. The chicken yolk sac IgY receptor (FcRY) is the ortholog of the mammalian phospholipase A2 receptor, a mannose receptor family member, rather than an FcRn or MHC homolog. FcRn and FcRY both exhibit ligand binding at the acidic pH of endosomes and ligand release at the slightly basic pH of blood. Here we show that FcRY expressed in polarized mammalian epithelial cells functioned in endocytosis, bidirectional transcytosis, and recycling of chicken FcY/IgY. Confocal immunofluorescence studies demonstrated that IgY binding and endocytosis occurred at acidic but not basic pH, mimicking pH-dependent uptake of IgG by FcRn. Colocalization studies showed FcRY-mediated internalization via clathrin-coated pits and transport involving early and recycling endosomes. Disruption of microtubules partially inhibited apical-to-basolateral and basolateral-to-apical transcytosis, but not recycling, suggesting the use of different trafficking machinery. Our results represent the first cell biological evidence of functional equivalence between FcRY and FcRn and provide an intriguing example of how evolution can give rise to systems in which similar biological requirements in different species are satisfied utilizing distinct protein folds.


Asunto(s)
Inmunoglobulinas/metabolismo , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Fc/metabolismo , Saco Vitelino/inmunología , Animales , Línea Celular , Polaridad Celular , Embrión de Pollo , Endocitosis/inmunología , Células Epiteliales/citología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunización Pasiva , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/inmunología , Túbulos Renales Colectores/metabolismo , Lectinas Tipo C/genética , Receptor de Manosa , Lectinas de Unión a Manosa/genética , Ratas , Receptores de Superficie Celular/genética , Receptores Fc/genética , Receptores de Fosfolipasa A2/genética , Receptores de Fosfolipasa A2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Saco Vitelino/metabolismo
16.
J Exp Med ; 204(12): 2837-52, 2007 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-17967904

RESUMEN

Ascending urinary tract infection (UTI) and pyelonephritis caused by uropathogenic Escherichia coli (UPEC) are very common infections that can cause severe kidney damage. Collecting duct cells, the site of hormonally regulated ion transport and water absorption controlled by vasopressin, are the preferential intrarenal site of bacterial adhesion and initiation of inflammatory response. We investigated the effect of the potent V2 receptor (V2R) agonist deamino-8-D-arginine vasopressin (dDAVP) on the activation of the innate immune response using established and primary cultured collecting duct cells and an experimental model of ascending UTI. dDAVP inhibited Toll-like receptor 4-mediated nuclear factor kappaB activation and chemokine secretion in a V2R-specific manner. The dDAVP-mediated suppression involved activation of protein phosphatase 2A and required an intact cystic fibrosis transmembrane conductance regulator Cl- channel. In vivo infusion of dDAVP induced a marked fall in proinflammatory mediators and neutrophil recruitment, and a dramatic rise in the renal bacterial burden in mice inoculated with UPECs. Conversely, administration of the V2R antagonist SR121463B to UPEC-infected mice stimulated both the local innate response and the antibacterial host defense. These findings evidenced a novel hormonal regulation of innate immune cellular activation and demonstrate that dDAVP is a potent modulator of microbial-induced inflammation in the kidney.


Asunto(s)
Arginina Vasopresina/fisiología , Túbulos Renales Colectores/fisiología , Receptor Toll-Like 4/fisiología , Infecciones Urinarias/inmunología , Adulto , Animales , Niño , Infecciones por Escherichia coli/inmunología , Humanos , Trasplante de Riñón , Túbulos Renales Colectores/inmunología , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Transducción de Señal , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Urotelio/inmunología , Urotelio/fisiología
17.
J Immunol ; 177(7): 4773-84, 2006 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-16982918

RESUMEN

TLR4 plays a central role in resistance to pyelonephritis caused by uropathogenic Escherichia coli (UPEC). It has been suggested that renal tubule epithelial cells expressing TLRs may play a key role in inflammatory disorders and in initiating host defenses. In this study we used an experimental mouse model of ascending urinary tract infection to show that UPEC isolates preferentially adhered to the apical surface of medullary collecting duct (MCD) intercalated cells. UPEC-infected C3H/HeJ (Lps(d)) mice carrying an inactivating mutation of tlr4 failed to clear renal bacteria and exhibited a dramatic slump in proinflammatory mediators as compared with infected wild-type C3H/HeOuJ (Lps(n)) mice. However, the level of expression of the leukocyte chemoattractants MIP-2 and TNF-alpha still remained greater in UPEC-infected than in naive C3H/HeJ (Lps(d)) mice. Using primary cultures of microdissected Lps(n) MCDs that expressed TLR4 and its accessory molecules MD2, MyD88, and CD14, we also show that UPECs stimulated both a TLR4-mediated, MyD88-dependent, TIR domain-containing adaptor-inducing IFN-beta-independent pathway and a TLR4-independent pathway, leading to bipolarized secretion of MIP-2. Stimulation by UPECs of the TLR4-mediated pathway in Lps(n) MCDs leads to the activation of NF-kappaB, and MAPK p38, ERK1/2, and JNK. In addition, UPECs stimulated TLR4-independent signaling by activating a TNF receptor-associated factor 2-apoptosis signal-regulatory kinase 1-JNK pathway. These findings demonstrate that epithelial collecting duct cells are actively involved in the initiation of an immune response via several distinct signaling pathways and suggest that intercalated cells play an active role in the recognition of UPECs colonizing the kidneys.


Asunto(s)
Células Epiteliales/microbiología , Escherichia coli/inmunología , Túbulos Renales Colectores/microbiología , Pielonefritis/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunología , Animales , Western Blotting , Quimiocinas/biosíntesis , Quimiocinas/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino , Humanos , Immunoblotting , Inflamación/inmunología , Inflamación/microbiología , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/inmunología , Ratones , Ratones Mutantes , Microscopía Electrónica de Rastreo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Pielonefritis/microbiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4/metabolismo , Infecciones Urinarias/complicaciones
18.
J Immunol ; 164(11): 5866-70, 2000 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-10820266

RESUMEN

Lung surfactant protein-D (SP-D), a collectin mainly produced by alveolar type II cells, initiates the effector mechanisms of innate immunity on binding to microbial carbohydrates. A panel of mRNAs from human tissues was screened for SP-D mRNA by RT-PCR. The lung was the main site of synthesis, but transcripts were readily amplified from trachea, brain, testis, salivary gland, heart, prostate gland, kidney, and pancreas. Minor sites of synthesis were uterus, small intestine, placenta, mammary gland, and stomach. The sequence of SP-D derived from parotid gland mRNA was identical with that of pulmonary SP-D. mAbs were raised against SP-D, and one was used to locate SP-D in cells and tissues by immunohistochemistry. SP-D immunoreactivity was found in alveolar type II cells, Clara cells, on and within alveolar macrophages, in epithelial cells of large and small ducts of the parotid gland, sweat glands, and lachrymal glands, in epithelial cells of the gall bladder and intrahepatic bile ducts, and in exocrine pancreatic ducts. SP-D was also present in epithelial cells of the skin, esophagus, small intestine, and urinary tract, as well as in the collecting ducts of the kidney. SP-D is generally present on mucosal surfaces and not restricted to a subset of cells in the lung. The localization and functions of SP-D indicate that this collectin is the counterpart in the innate immune system of IgA in the adaptive immune system.


Asunto(s)
Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Surfactantes Pulmonares/inmunología , Surfactantes Pulmonares/metabolismo , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Epidermis/química , Epidermis/inmunología , Epitelio/química , Epitelio/inmunología , Glándulas Exocrinas/química , Glándulas Exocrinas/inmunología , Glicoproteínas/biosíntesis , Glicoproteínas/química , Humanos , Inmunidad Mucosa , Inmunohistoquímica , Mucosa Intestinal/química , Mucosa Intestinal/inmunología , Túbulos Renales Colectores/química , Túbulos Renales Colectores/inmunología , Pulmón/química , Pulmón/inmunología , Membrana Mucosa/inmunología , Membrana Mucosa/metabolismo , Especificidad de Órganos/inmunología , Proteína D Asociada a Surfactante Pulmonar , Surfactantes Pulmonares/biosíntesis , Surfactantes Pulmonares/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
19.
Oncogene ; 19(10): 1288-96, 2000 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-10713670

RESUMEN

Prostate stem cell antigen (PSCA) is a recently defined homologue of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA mRNA is expressed in the basal cells of normal prostate and in more than 80% of prostate cancers. The purpose of the present study was to examine PSCA protein expression in clinical specimens of human prostate cancer. Five monoclonal antibodies were raised against a PSCA-GST fusion protein and screened for their ability to recognize PSCA on the cell surface of human prostate cancer cells. Immunohistochemical analysis of PSCA expression was performed on paraffin-embedded sections from 25 normal tissues, 112 primary prostate cancers and nine prostate cancers metastatic to bone. The level of PSCA expression in prostate tumors was quantified and compared with expression in adjacent normal glands. The antibodies detect PSCA expression on the cell surface of normal and malignant prostate cells and distinguish three extracellular epitopes on PSCA. Prostate and transitional epithelium reacted strongly with PSCA. PSCA staining was also seen in placental trophoblasts, renal collecting ducts and neuroendocrine cells in the stomach and colon. All other normal tissues tested were negative. PSCA protein expression was identified in 105/112 (94%) primary prostate tumors and 9/9 (100%) bone metastases. The level of PSCA expression increased with higher Gleason score (P=0.016), higher tumor stage (P=0.010) and progression to androgen-independence (P=0. 021). Intense, homogeneous staining was seen in all nine bone metastases. PSCA is a cell surface protein with limited expression in extraprostatic normal tissues. PSCA expression correlates with tumor stage, grade and androgen independence and may have prognostic utility. Because expression on the surface of prostate cancer cells increases with tumor progression, PSCA may be a useful molecular target in advanced prostate cancer.


Asunto(s)
Antígenos de Neoplasias/aislamiento & purificación , Neoplasias Óseas/inmunología , Neoplasias Óseas/secundario , Glicoproteínas de Membrana/aislamiento & purificación , Proteínas de Neoplasias/aislamiento & purificación , Neoplasias de la Próstata/inmunología , Anticuerpos Monoclonales , Anticuerpos Antineoplásicos , Antígenos de Neoplasias/inmunología , Sistema Digestivo/inmunología , Epítopos , Proteínas Ligadas a GPI , Humanos , Túbulos Renales Colectores/inmunología , Masculino , Glicoproteínas de Membrana/inmunología , Proteínas de Neoplasias/inmunología , Estadificación de Neoplasias , Sistemas Neurosecretores/inmunología , Neoplasias de la Próstata/patología , Distribución Tisular , Trofoblastos/inmunología
20.
Am J Physiol ; 275(2): F183-90, 1998 08.
Artículo en Inglés | MEDLINE | ID: mdl-9691006

RESUMEN

The collecting duct of the renal tubule contains two cell types, one of which, the intercalated cell, is responsible for acidification and alkalinization of urine. These cells exist in a multiplicity of morphological forms, with two extreme types, alpha and beta. The former acidifies the urine by an apical proton-translocating ATPase and a basolateral Cl/HCO3 exchanger, which is an alternately spliced form of band 3. This kidney form of band 3, kAE1, is present in the apical membrane of the beta-cell, which has the H+-ATPase on the basolateral membrane. We had suggested previously that metabolic acidosis leads to conversion of beta-types to alpha-types. To study the biochemical basis of this plasticity, we used an immortalized cell line of the beta-cell and showed that these cells convert to the alpha-phenotype when plated at superconfluent density. At high density these cells localize a new protein, which we term "hensin," to the extracellular matrix, and hensin acts as a molecular switch capable of changing the phenotype of these cells in vitro. Hensin induces new cytoskeletal proteins, makes the cells assume a more columnar shape and retargets kAE1 and the H+-ATPase. These recent studies suggest that the conversion of beta- to alpha-cells, at least in vitro, bears many of the hallmarks of terminal differentiation.


Asunto(s)
Túbulos Renales Colectores/citología , Túbulos Renales Colectores/fisiología , Proteínas de la Membrana , Receptores Inmunológicos/fisiología , Receptores de Lipoproteína , Animales , Membrana Celular/fisiología , Proteínas del Citoesqueleto/biosíntesis , Humanos , Concentración de Iones de Hidrógeno , Túbulos Renales Colectores/inmunología , Fenotipo , ATPasas de Translocación de Protón/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase B , Orina/fisiología
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