RESUMEN
Renal medullary carcinomas (RMCs) and collecting duct carcinomas (CDCs) are rare subsets of lethal high-stage, high-grade distal nephron-related adenocarcinomas with a predilection for the renal medullary region. Recent findings have established an emerging group of fumarate hydratase (FH)-deficient tumors related to hereditary leiomyomatosis and renal cell carcinoma (HLRCC-RCCs) syndrome within this morphologic spectrum. Recently developed, reliable ancillary testing has enabled consistent separation between these tumor types. Here, we present the clinicopathologic features and differences in the morphologic patterns between RMC, CDC, and FH-deficient RCC in consequence of these recent developments. This study included a total of 100 cases classified using contemporary criteria and ancillary tests. Thirty-three RMCs (SMARCB1/INI1-deficient, hemoglobinopathy), 38 CDCs (SMARCB1/INI1-retained), and 29 RCCs defined by the FH-deficient phenotype (FH/2SC or FH/2SC with FH mutation, regardless of HLRCC syndromic stigmata/history) were selected. The spectrum of morphologic patterns was critically evaluated, and the differences between the morphologic patterns present in the 3 groups were analyzed statistically. Twenty-five percent of cases initially diagnosed as CDC were reclassified as FH-deficient RCC on the basis of our contemporary diagnostic approach. Among the different overlapping morphologic patterns, sieve-like/cribriform and reticular/yolk sac tumor-like patterns favored RMCs, whereas intracystic papillary and tubulocystic patterns favored FH-deficient RCC. The tubulopapillary pattern favored both CDCs and FH-deficient RCCs, and the multinodular infiltrating papillary pattern favored CDCs. Infiltrating glandular and solid sheets/cords/nested patterns were not statistically different among the 3 groups. Viral inclusion-like macronucleoli, considered as a hallmark of HLRCC-RCCs, were observed significantly more frequently in FH-deficient RCCs. Despite the overlapping morphology found among these clinically aggressive infiltrating high-grade adenocarcinomas of the kidney, reproducible differences in morphology emerged between these categories after rigorous characterization. Finally, we recommend that definitive diagnosis of CDC should only be made if RMC and FH-deficient RCC are excluded.
Asunto(s)
Biomarcadores de Tumor/deficiencia , Carcinoma de Células Renales/patología , Fumarato Hidratasa/deficiencia , Médula Renal/patología , Neoplasias Renales/patología , Túbulos Renales Colectores/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Australia , Biomarcadores de Tumor/genética , Biopsia , Brasil , Canadá , Carcinoma de Células Renales/clasificación , Carcinoma de Células Renales/enzimología , Carcinoma de Células Renales/genética , Niño , Análisis Mutacional de ADN , Diagnóstico Diferencial , Europa (Continente) , Femenino , Fumarato Hidratasa/genética , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Médula Renal/enzimología , Neoplasias Renales/clasificación , Neoplasias Renales/enzimología , Neoplasias Renales/genética , Túbulos Renales Colectores/enzimología , Masculino , Persona de Mediana Edad , Mutación , Clasificación del Tumor , Fenotipo , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Estados Unidos , Adulto JovenRESUMEN
Epithelial tissue requires that cells attach to each other and to the extracellular matrix by the assembly of adherens junctions (AJ) and focal adhesions (FA) respectively. We have previously shown that, in renal papillary collecting duct (CD) cells, both AJ and FA are located in sphingomyelin (SM)-enriched plasma membrane microdomains. In the present work, we investigated the involvement of SM metabolism in the preservation of the epithelial cell phenotype and tissue organization. To this end, primary cultures of renal papillary CD cells were performed. Cultured cells preserved the fully differentiated epithelial phenotype as reflected by the presence of primary cilia. Cells were then incubated for 24h with increasing concentrations of D609, a SM synthase (SMS) inhibitor. Knock-down experiments silencing SMS 1 and 2 were also performed. By combining biochemical and immunofluorescence studies, we found experimental evidences suggesting that, in CD cells, SMS 1 activity is essential for the preservation of cell-cell adhesion structures and therefore for the maintenance of CD tissue/tubular organization. The inhibition of SMS 1 activity induced CD cells to lose their epithelial phenotype and to undergo an epithelial-mesenchymal transition (EMT) process.
Asunto(s)
Células Epiteliales/enzimología , Transición Epitelial-Mesenquimal , Túbulos Renales Colectores/enzimología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/antagonistas & inhibidores , Animales , Adhesión Celular , Células Epiteliales/citología , Túbulos Renales Colectores/citología , Masculino , Ratas , Ratas Wistar , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
The objective of the present work was to characterize the biochemical activity of the proton pumps present in the C11 clone of Madin-Darby canine kidney (MDCK) cells, akin to intercalated cells of the collecting duct, as well as to study their regulation by hormones like aldosterone and vasopressin. MDCK-C11 cells from passages 78 to 86 were utilized. The reaction to determine H+-ATPase activity was started by addition of cell homogenates to tubes contained the assay medium. The inorganic phosphate (P(i)) released was determined by a colorimetric method modified from that described by Fiske and Subbarow. Changes in intracellular calcium concentration in the cells was determined using the Ca2+-sensing dye fluo-4 AM. Homogenates of MDCK-C11 cells present a bafilomycin-sensitive activity (vacuolar H+-ATPase), and a vanadate-sensitive activity (H+/K+-ATPase). The bafilomycin-sensitive activity showed a pH optimum of 6.12. ATPase activity was also stimulated in a dose-dependent fashion as K+ concentration was increased between 0 and 50 mmol x L(-1), with an apparent K(m) for the release of P(i) of 0.13 mmol x L(-1) and Vmax of 22.01 nmol x mg(-1) x min(-1). Incubation of cell monolayers with 10(-8) mol x L(-1) aldosterone for 24 h significantly increased vacuolar H+-ATPase activity, an effect prevented by 10(-5) mol x L(-1) spironolactone. Vacuolar H+-ATPase activity was also stimulated by 10(-11) mol x L(-1) vasopressin, an effect prevented by a V1 receptor-specific antagonist. This dose of vasopressin determined a sustained rise of cytosolic ionized calcium. We conclude that (i) homogenates of MDCK-C11 cells present a bafilomycin-sensitive (H+-ATPase) activity and a vanadate-sensitive (H+/K+-ATPase) activity, and (ii) vacuolar H+-ATPase activity is activated by aldosterone through a genomic pathway and by vasopressin through V1 receptors.
Asunto(s)
Aldosterona/farmacología , ATPasas de Translocación de Protón/metabolismo , Vasopresinas/farmacología , Aldosterona/fisiología , Animales , Calcio/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Perros , Concentración de Iones de Hidrógeno , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/enzimología , Cloruro de Potasio/farmacología , Vasopresinas/fisiologíaRESUMEN
We described in mouse inner medullary-collecting duct cells (mIMCD-3) the somatic and the N-domain ACE synthesis and its interaction with the kallikrein-kinin system co-localized in the same cells. We purified two ACE forms from culture medium, M1 (130 kDa) and M2 (N-domain, 60 kDa), and cellular lysate, C1 (130 kDa) and C2 (N-domain, 60 kDa). Captopril and enalaprilat inhibited the purified enzymes. The immunofluorescence studies indicated that ACE is present in the membrane, cytoplasm and in the cell nucleus. Kinin B1 and B2 receptors were detected by immunofluorescence and showed to be activated by BK and DesR9 BK, increasing the acidification rate which was enhanced in the presence of enalaprilat. The presence of secreted and intracellular ACE in mIMCD-3 confirmed the hypothesis previously proposed by our group for a new site of ACE secretion in the collecting duct.
Asunto(s)
Médula Renal/enzimología , Túbulos Renales Colectores/enzimología , Peptidil-Dipeptidasa A/aislamiento & purificación , Receptor de Bradiquinina B2/análisis , Animales , Captopril/farmacología , Células Cultivadas , Enalaprilato/farmacología , Técnica del Anticuerpo Fluorescente , Ratones , Peptidil-Dipeptidasa A/análisisRESUMEN
The purpose of this study was to determine the basal levels of dopamine (DA) and to examine the enzymes involved in DA metabolism in different microdissected nephron segments from rat kidneys. Segments were incubated with DA (50 nM) or DA plus monoamine oxidase (MAO) or catechol-O-methyl transferase (COMT) inhibitors. Basal DA levels were higher in the proximal convoluted tubule (PCT, 10.8+/-3.7 pg/mm) and in the medullary collecting duct (MCD, 10.9+/-4.0 pg/mm) than in the medullary thick ascending limb of Henle's loop (MTAL, 4.9+/-0.9 pg/mm) (P<0.05). The percentage of exogenously added DA that was not metabolised was similar in both PCT (67+/-13%) and MCD (65+/-5%) and lower in MTAL (35+/-7%), suggesting that MTAL is a major site of DA metabolism. Inhibition of MAO (pargyline 1 mM) significantly increased the basal content of DA and the percentage of the added non-metabolised DA (to 95+/-10%) in PCT but had no effect on MTAL or MCD. Conversely, inhibition of COMT (nitecapone or Ro-41-0960, both 1 mM) slightly increased the basal levels of DA only in MTAL, whereas the percentage of added DA not metabolised rose to 97+/-10% in MTAL and to 91+/-15% in MCD. COMT inhibition had no effect in PCT. In conscious rats pargyline (50 mg/kg) increased urinary DA from 680+/-34 to 1,128+/-158 ng/d/100 g BW (P<0.01) while nitecapone (40 mg/kg) produced a slight non-significant increment. Our results show that DA is present all along the rat nephron and that renal DA is metabolised continuously and predominantly by MAO in proximal segments, and by COMT in the more distal ones.
Asunto(s)
Dopamina/metabolismo , Nefronas/enzimología , Animales , Benzofenonas/farmacología , Catecol O-Metiltransferasa/metabolismo , Inhibidores de Catecol O-Metiltransferasa , Catecoles/farmacología , Corteza Renal/enzimología , Médula Renal/enzimología , Túbulos Renales Colectores/enzimología , Túbulos Renales Proximales/enzimología , Asa de la Nefrona/enzimología , Masculino , Monoaminooxidasa/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Natriuresis/efectos de los fármacos , Pargilina/farmacología , Pentanonas/farmacología , Ratas , Ratas WistarRESUMEN
Chronic ethanol exposure causes alterations in biologic membranes of different cell types. (Na + K) adenosine triphosphatase (ATPase), a membrane-bound enzyme inhibited by the acute presence of ethanol, increases its activity in rat kidney after chronic ethanol consumption. The aim of this investigation was to evaluate the effect of ethanol on the modulation of (Na + K)-ATPase by glucocorticoids and mineralocorticoids in renal papillary collecting duct cells. Cultured renal papillary collecting duct cells were exposed to a medium containing 150 mM ethanol plus either 100 nM aldosterone or 10 nM dexamethasone. Control groups were cultured in the absence of ethanol and/or the hormones. Mg(2+)-ATPase was used as control enzyme. The activity of ATPases was measured by ATP hydrolysis. Ethanol increased the activities of (Na + K)-ATPase and Mg(2+)- ATPase in 29 and 33% of controls, respectively; only (Na + K)-ATPase activity was elevated in the presence of aldosterone or dexamethasone, whereas Mg(2+)-ATPase was unaltered by these hormones. The effects of aldosterone and dexamethasone on (Na + K)-ATPase activity were augmented by ethanol in 50 and 19% of controls, respectively. These results suggest that ethanol treatment enhances the upregulation of (Na + K)-ATPase activity by both aldosterone and dexamethasone, in cultured renal papillary collecting duct cells.
Asunto(s)
Aldosterona/farmacología , Dexametasona/farmacología , Etanol/farmacología , Médula Renal/enzimología , Túbulos Renales Colectores/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfato/metabolismo , Animales , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Células Cultivadas , Glucocorticoides/farmacología , Homeostasis/efectos de los fármacos , Médula Renal/efectos de los fármacos , Túbulos Renales Colectores/efectos de los fármacos , Masculino , Ratas , Ratas WistarRESUMEN
The luminal membrane of collecting duct cells, specially the intercalated cells, is normally exposed to active kallikrein. This is due to the specific localization of renal kallikrein in the connecting tubule cells. We have previously reported inhibition of distal bicarbonate secretion by renal kallikrein. The present study was performed to evaluate the participation of basolateral Cl-/HCO3- exchanger and luminal H(+)-ATPase activity of cortical collecting duct segments (CCD) in the mechanism involved in the inhibition of bicarbonate secretion induced by the enzyme. The effect of orthograde injections of 1 microgram/ml (250 U/6.3 mg) pig pancreatic kallikrein, in the absence and presence of 1 mM DIDS (stilbene-disulfonic acid) in the renal tubule system, was evaluated. Urine fractions were collected after two-minutes stop-flow. Changes in the urine fraction (Fr) related to those in free-flow urine samples (Ff) were related to the respective polyfructosan (Inutest) ratio. Renal kallikrein activity (Fr:Ff kallikrein/Fr:Ff polyfructosan) increased significantly in the first 120 microliters urine fraction collected after glandular 1 microgram/ml kallikrein, P < 0.05, (first stop-flow) and after glandular 1 microgram/ml kallikrein plus 1 mM. DIDS P < 0.05 (second stop flow). Bicarbonate secretion rate (Fr:Ff HCO3-/Fr:Ff polyfructosan) of collecting ducts was significantly reduced in the first 120 microliters urine fraction collected, related to control, during the first and second stop-flow periods. No difference was shown in bicarbonate excretion between the first 120 microliters urine fractions collected after administration of glandular kallikrein and glandular kallikrein plus DIDS. To measure H(+)-ATPase activity, rat microdissected cortical collector tubules (CCD) were incubated in the presence of increasing glandular kallikrein doses (A: 93, B: 187 and C: 375 mU/200 microL) in the presence of ouabain (4 microM) and omeprazole (100 microM) to inhibit Na(+)-K(+)-ATPase and H(+)-K(+)-ATPase, respectively. In CCD, bafilomycin-sensitive H(+)-ATPase activity (pmol/mm/min) after increasing kallikrein doses did not differ significantly from control...(AU)
Asunto(s)
Animales , Femenino , Ratas , Antiportadores/metabolismo , Bicarbonatos/metabolismo , Transporte Biológico , Antiportadores de Cloruro-Bicarbonato , Coagulantes/farmacología , Calicreínas/farmacología , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/enzimología , ATPasas de Translocación de Protón/metabolismo , Ratas Endogámicas WKYRESUMEN
The luminal membrane of collecting duct cells, specially the intercalated cells, is normally exposed to active kallikrein. This is due to the specific localization of renal kallikrein in the connecting tubule cells. We have previously reported inhibition of distal bicarbonate secretion by renal kallikrein. The present study was performed to evaluate the participation of basolateral Cl-/HCO3- exchanger and luminal H(+)-ATPase activity of cortical collecting duct segments (CCD) in the mechanism involved in the inhibition of bicarbonate secretion induced by the enzyme. The effect of orthograde injections of 1 microgram/ml (250 U/6.3 mg) pig pancreatic kallikrein, in the absence and presence of 1 mM DIDS (stilbene-disulfonic acid) in the renal tubule system, was evaluated. Urine fractions were collected after two-minutes stop-flow. Changes in the urine fraction (Fr) related to those in free-flow urine samples (Ff) were related to the respective polyfructosan (Inutest) ratio. Renal kallikrein activity (Fr:Ff kallikrein/Fr:Ff polyfructosan) increased significantly in the first 120 microliters urine fraction collected after glandular 1 microgram/ml kallikrein, P < 0.05, (first stop-flow) and after glandular 1 microgram/ml kallikrein plus 1 mM. DIDS P < 0.05 (second stop flow). Bicarbonate secretion rate (Fr:Ff HCO3-/Fr:Ff polyfructosan) of collecting ducts was significantly reduced in the first 120 microliters urine fraction collected, related to control, during the first and second stop-flow periods. No difference was shown in bicarbonate excretion between the first 120 microliters urine fractions collected after administration of glandular kallikrein and glandular kallikrein plus DIDS. To measure H(+)-ATPase activity, rat microdissected cortical collector tubules (CCD) were incubated in the presence of increasing glandular kallikrein doses (A: 93, B: 187 and C: 375 mU/200 microL) in the presence of ouabain (4 microM) and omeprazole (100 microM) to inhibit Na(+)-K(+)-ATPase and H(+)-K(+)-ATPase, respectively. In CCD, bafilomycin-sensitive H(+)-ATPase activity (pmol/mm/min) after increasing kallikrein doses did not differ significantly from control...
Asunto(s)
Animales , Femenino , Ratas , Antiportadores , ATPasas de Translocación de Protón/metabolismo , Bicarbonatos , Transporte Biológico , Calicreínas/farmacología , Antiportadores de Cloruro-Bicarbonato , Coagulantes , Ratas Endogámicas WKY , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/enzimologíaRESUMEN
The luminal membrane of collecting duct cells, specially the intercalated cells, is normally exposed to active kallikrein. This is due to the specific localization of renal kallikrein in the connecting tubule cells. We have previously reported inhibition of distal bicarbonate secretion by renal kallikrein. The present study was performed to evaluate the participation of basolateral Cl-/HCO3- exchanger and luminal H(+)-ATPase activity of cortical collecting duct segments (CCD) in the mechanism involved in the inhibition of bicarbonate secretion induced by the enzyme. The effect of orthograde injections of 1 microgram/ml (250 U/6.3 mg) pig pancreatic kallikrein, in the absence and presence of 1 mM DIDS (stilbene-disulfonic acid) in the renal tubule system, was evaluated. Urine fractions were collected after two-minutes stop-flow. Changes in the urine fraction (Fr) related to those in free-flow urine samples (Ff) were related to the respective polyfructosan (Inutest) ratio. Renal kallikrein activity (Fr:Ff kallikrein/Fr:Ff polyfructosan) increased significantly in the first 120 microliters urine fraction collected after glandular 1 microgram/ml kallikrein, P < 0.05, (first stop-flow) and after glandular 1 microgram/ml kallikrein plus 1 mM. DIDS P < 0.05 (second stop flow). Bicarbonate secretion rate (Fr:Ff HCO3-/Fr:Ff polyfructosan) of collecting ducts was significantly reduced in the first 120 microliters urine fraction collected, related to control, during the first and second stop-flow periods. No difference was shown in bicarbonate excretion between the first 120 microliters urine fractions collected after administration of glandular kallikrein and glandular kallikrein plus DIDS. To measure H(+)-ATPase activity, rat microdissected cortical collector tubules (CCD) were incubated in the presence of increasing glandular kallikrein doses (A: 93, B: 187 and C: 375 mU/200 microL) in the presence of ouabain (4 microM) and omeprazole (100 microM) to inhibit Na(+)-K(+)-ATPase and H(+)-K(+)-ATPase, respectively. In CCD, bafilomycin-sensitive H(+)-ATPase activity (pmol/mm/min) after increasing kallikrein doses did not differ significantly from control. No difference related to control H(+)-ATPase activity was observed when microdissected CCD segments were incubated in the presence of an AT1 receptor antagonist (Losartan 10(-6) M) and glandular kallikrein (93 mU). On the contrary, angiotensin II (10(-8) M) significantly decreased H(+)-ATPase activity. The present study shows that neither basolateral Cl-/HCO3- exchanger nor H(+)-ATPase activity are involved in bicarbonate inhibition by glandular kallikrein at CCD. Involvement of luminal Cl-/HCO3- exchanger at beta intercalated cells in CCD may be suggested for the bicarbonate secretion inhibition induced by renal kallikrein.
Asunto(s)
Antiportadores/metabolismo , Bicarbonatos/metabolismo , Coagulantes/farmacología , Calicreínas/farmacología , Túbulos Renales Colectores/enzimología , ATPasas de Translocación de Protón/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Antiportadores de Cloruro-Bicarbonato , Femenino , Túbulos Renales Colectores/citología , Ratas , Ratas Endogámicas WKYRESUMEN
The activity of angiotensin I-converting enzyme (ACE) was determined in tubular fluid collected from several portions of the rat nephron and urine and in total and efferent arteriolar blood using hippuryl-L-His-Leu as substrate. ACE activity decreased 30% from the pre- to the postglomerular arterioles (P < 0.001), suggesting a role of the glomerulus in ACE clearance. The enzyme activity was found to be present throughout the rat nephron. However, the highest activities were found in the proximal tubule and urine (0.692 +/- 0.007 and 1.05 +/- 0.015 pmol x microl(-1) x min(-1), respectively). Compared with other segments, ACE activity decreased from the initial portion of the proximal tubule to the distal nephron and increased again in the urine. Along the proximal tubule, ACE was secreted and degraded and/or reabsorbed and then secreted again into the collecting duct; no ACE activity was found in the late distal tubule, but a high level was detected in the urine, indicating a potential physiological role in the inactivation of the kinins formed by kallikrein beyond the connecting tubules. Moreover, the possible role of mesangial cells (MC) in the decrease of intraglomerular ACE was also evaluated. The analysis of ACE gene showed that MC in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on MC function.
Asunto(s)
Mesangio Glomerular/enzimología , Túbulos Renales/enzimología , Nefronas/enzimología , Peptidil-Dipeptidasa A/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Mesangio Glomerular/citología , Túbulos Renales Colectores/enzimología , Túbulos Renales Proximales/enzimología , Masculino , Oligopéptidos , Peptidil-Dipeptidasa A/orina , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
This study was designed to measure H-K-ATPase and Na-K-ATPase activities in rat inner medullary collecting duct (IMCD) cells during potassium depletion and loading. Both enzyme activities were similarly distributed from the proximal to the distal portion of this nephron segment. One week K depletion did not stimulate H-K-ATPase activity but reduced Na-K-ATPase activity. Within 2 weeks after the onset of potassium depletion, the rats developed metabolic alkalosis, and H-K-ATPase activity was suppressed while Na-K-ATPase activity had returned to control values. H-K-ATPase activity was suppressed following potassium loading whereas Na-K-ATPase activity was unchanged. The results are consistent with the presence of H-K-ATPase in IMCD and indicate that H-K-ATPase and Na-K-ATPase activities are modulated by potassium intake.