RESUMO
Gestational potassium retention, most of which occurs during late pregnancy, is essential for fetal development. The purpose of this study was to examine mechanisms underlying changes in potassium handling by the kidney and colon in pregnancy. We found that potassium intake and renal excretion increased in late pregnancy while fecal potassium excretion remained unchanged and that pregnant rats exhibited net potassium retention. By quantitative PCR we found markedly increased H+-K+-ATPase type 2 (HKA2) mRNA expression in the cortex and outer medullary of late pregnant vs. virgin. Renal outer medullary potassium channel (ROMK) mRNA was unchanged in the cortex, but apical ROMK abundance (by immunofluorescence) was decreased in pregnant vs. virgin in the distal convoluted tubule (DCT) and connecting tubule (CNT). Big potassium-α (BKα) channel-α protein abundance in intercalated cells in the cortex and outer medullary collecting ducts (by immunohistochemistry) fell in late pregnancy. In the distal colon we found increased HKA2 mRNA and protein abundance (Western blot) and decreased BKα protein with no observed changes in mRNA. Therefore, the potassium retention of pregnancy is likely to be due to increased collecting duct potassium reabsorption (via increased HKA2), decreased potassium secretion (via decreased ROMK and BK), as well as increased colonic reabsorption via HKA2.
Assuntos
Colo/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Túbulos Renais Coletores/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Potássio/metabolismo , Animais , Transporte Biológico , Feminino , Idade Gestacional , ATPase Trocadora de Hidrogênio-Potássio/genética , Reabsorção Intestinal , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Potássio/sangue , Potássio/urina , Canais de Potássio Corretores do Fluxo de Internalização/genética , Gravidez , Ratos Sprague-Dawley , Eliminação Renal , Reabsorção RenalRESUMO
ROMK channels are well-known to play a central role in renal K secretion, but the absence of highly specific and avid-ROMK antibodies has presented significant roadblocks toward mapping the extent of expression along the entire distal nephron and determining whether surface density of these channels is regulated in response to physiological stimuli. Here, we prepared new ROMK antibodies verified to be highly specific, using ROMK knockout mice as a control. Characterization with segmental markers revealed a more extensive pattern of ROMK expression along the entire distal nephron than previously thought, localizing to distal convoluted tubule regions, DCT1 and DCT2; the connecting tubule (CNT); and cortical collecting duct (CD). ROMK was diffusely distributed in intracellular compartments and at the apical membrane of each tubular region. Apical labeling was significantly increased by high-K diet in DCT2, CNT1, CNT2, and CD (P < 0.05) but not in DCT1. Consistent with the large increase in apical ROMK, dramatically increased mature glycosylation was observed following dietary potassium augmentation. We conclude 1) our new antibody provides a unique tool to characterize ROMK channel localization and expression and 2) high-K diet causes a large increase in apical expression of ROMK in DCT2, CNT, and CD but not in DCT1, indicating that different regulatory mechanisms are involved in K diet-regulated ROMK channel functions in the distal nephron.
Assuntos
Néfrons/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Potássio na Dieta/metabolismo , Análise de Variância , Animais , Western Blotting , Camundongos , Camundongos Knockout , Canais de Potássio Corretores do Fluxo de Internalização/genéticaRESUMO
Apical SK/ROMK and BK channels mediate baseline and flow-induced K secretion (FIKS), respectively, in the cortical collecting duct (CCD). BK channels are detected in acid-base transporting intercalated (IC) and Na-absorbing principal (PC) cells. Although the density of BK channels is greater in IC than PC, Na-K-ATPase activity in IC is considered inadequate to sustain high rates of urinary K secretion. To test the hypothesis that basolateral NKCC in the CCD contributes to BK channel-mediated FIKS, we measured net K secretion (J(K)) and Na absorption (J(Na)) at slow (â¼1) and fast (â¼5 nl·min(-1)·mm(-1)) flow rates in rabbit CCDs microperfused in vitro in the absence and presence of bumetanide, an inhibitor of NKCC, added to the bath. Bumetanide inhibited FIKS but not basal J(K), J(Na), or the flow-induced [Ca(2+)](i) transient necessary for BK channel activation. Addition of luminal iberiotoxin, a BK channel inhibitor, to bumetanide-treated CCDs did not further reduce J(K). Basolateral Cl removal reversibly inhibited FIKS but not basal J(K) or J(Na). Quantitative PCR performed on single CCD samples using NKCC1- and 18S-specific primers and probes and the TaqMan assay confirmed the presence of the transcript in this nephron segment. To identify the specific cell type to which basolateral NKCC is localized, we exploited the ability of NKCC to accept NH(4)(+) at its K-binding site to monitor the rate of bumetanide-sensitive cytosolic acidification after NH(4)(+) addition to the bath in CCDs loaded with the pH indicator dye BCECF. Both IC and PC were found to have a basolateral bumetanide-sensitive NH(4)(+) entry step and NKCC1-specific antibodies labeled the basolateral surfaces of both cell types in CCDs. These results suggest that BK channel-mediated FIKS is dependent on a basolateral bumetanide-sensitive, Cl-dependent transport pathway, proposed to be NKCC1, in both IC and PC in the CCD.
Assuntos
Córtex Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Potássio/metabolismo , Simportadores de Cloreto de Sódio-Potássio/fisiologia , Animais , Bumetanida/farmacologia , Cálcio/metabolismo , Cátions/metabolismo , Diuréticos/farmacologia , Corantes Fluorescentes , Imuno-Histoquímica , Córtex Renal/citologia , Túbulos Renais Coletores/citologia , Masculino , Néfrons/metabolismo , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Compostos de Amônio Quaternário/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Coelhos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simportadores de Cloreto de Sódio-Potássio/genética , Membro 2 da Família 12 de Carreador de SolutoRESUMO
We have discovered that the immunoreactivity of the fluorophore Alexa Fluor 488 survives glutaraldehyde and osmium tetroxide fixation and epoxy resin embedding and etching. We have developed new localization methods that for the first time take advantage of this property. The antigen is localized in cryosections using suitable primary antibody and an Alexa Fluor 488-conjugated secondary antibody. Cryosection fluorescence can be photographed for later correlation with electron microscopy (EM) findings. The sections are then further fixed with glutaraldehyde and OsO4, if desired and flat-embedded in epoxy resin. Semi-thin sections are etched completely with sodium ethoxide, whereas thin sections are partially etched. Alexa Fluor 488 is then localized with rabbit anti-Alexa Fluor 488 and goat anti-rabbit conjugated to Alexa Fluor 488 [light microscopy (LM)] or to colloidal gold (EM). A second antigen may also be localized using Alexa Fluor 568. When used without postfixation, these methods produce high-resolution semi-thin, or even thin, sections that retain a high level of fluorescence for LM observations. These methods allow highly sensitive immunolocalizations in tissue while preserving cell fine structure through traditional fixation and epoxy embedding. In demonstration of the methods, we describe the localization of the thiazide-sensitive sodium/chloride cotransporter and the epithelial sodium channel in rat kidney.
Assuntos
Anticorpos , Corantes Fluorescentes , Imuno-Histoquímica/métodos , Receptores de Droga/metabolismo , Canais de Sódio/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , Animais , Anticorpos/química , Canais Epiteliais de Sódio , Resinas Epóxi , Fixadores , Secções Congeladas , Glutaral , Cabras , Rim/metabolismo , Tetróxido de Ósmio , Inclusão em Plástico , Coelhos , Ratos , Ratos Sprague-Dawley , Sensibilidade e EspecificidadeRESUMO
A functional collaboration between growth factor receptors such as platelet derived growth factor receptor (PDGFR) and integrins is required for effective signal transduction in response to soluble growth factors. However, the mechanisms of synergistic PDGFR/integrin signaling remain poorly understood. Our previous work showed that cell surface tissue transglutaminase (tTG) induces clustering of integrins and amplifies integrin signaling by acting as an integrin binding adhesion co-receptor for fibronectin. Here we report that in fibroblasts tTG enhances PDGFR-integrin association by interacting with PDGFR and bridging the two receptors on the cell surface. The interaction between tTG and PDGFR reduces cellular levels of the receptor by accelerating its turnover. Moreover, the association of PDGFR with tTG causes receptor clustering, increases PDGF binding, promotes adhesion-mediated and growth factor-induced PDGFR activation, and up-regulates downstream signaling. Importantly, tTG is required for efficient PDGF-dependent proliferation and migration of fibroblasts. These results reveal a previously unrecognized role for cell surface tTG in the regulation of the joint PDGFR/integrin signaling and PDGFR-dependent cell responses.
Assuntos
Fibroblastos/citologia , Fibroblastos/enzimologia , Integrinas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transglutaminases/metabolismo , Animais , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Derme/citologia , Regulação para Baixo/fisiologia , Humanos , Camundongos , Células NIH 3T3 , Agregação de Receptores/fisiologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Transglutaminases/genética , Regulação para Cima/fisiologia , Cicatrização/fisiologiaRESUMO
Sodium-hydrogen exchanger regulatory factor-1 and -2 (NHERF-1 and NHERF-2) are adaptor proteins that regulate renal electrolyte transport and interact with the platelet-derived growth factor receptors (PDGFR). The distribution of the NHERF proteins and PDGFR was studied in normal human kidneys and in renal transplant rejection using immunocytochemistry. In normal kidneys, NHERF-1 was detected in proximal tubules. NHERF-2 was detected in glomeruli, peritubular capillaries, and collecting duct principal cells. NHERF-2 was also weakly detected in the proximal tubule. PDGFR-beta was detected in glomeruli but not in tubules while PDGFR-alpha was detected in renal tubules and minimally in glomeruli. Acute and chronic transplant rejection was associated with increased expression of PDGFR-alpha in tubules and expression in the glomeruli. PDGFR-beta expression in the glomeruli was increased in transplant rejection and became detectable in tubules. Expression of NHERF-1 and NHERF-2 was not different in the patient groups. These results indicate that in contrast to the rat, both NHERF isoforms are detected in the human proximal tubule. In renal transplant rejection, there is increased expression of both PDGFR subtypes consistent with a role for PDGF in injury or repair.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Rejeição de Enxerto/metabolismo , Transplante de Rim , Rim/metabolismo , Fosfoproteínas/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Doença Aguda , Doença Crônica , Técnica Indireta de Fluorescência para Anticorpo , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Transplante de Rim/imunologia , Transplante de Rim/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Trocadores de Sódio-HidrogênioRESUMO
In expression systems and in yeast, Na/H exchanger regulatory factor (NHERF)-1 and NHERF-2 have been demonstrated to interact with the renal brush border membrane proteins NHE3 and Npt2. In renal tissue of mice, however, NHERF-1 is required for cAMP regulation of NHE3 and for the apical targeting of Npt2 despite the presence of NHERF-2, suggesting another order of specificity. The present studies examine the subcellular location of NHERF-1 and NHERF-2 and their interactions with target proteins including NHE3, Npt2, and ezrin. The wild-type mouse proximal tubule expresses both NHERF-1 and NHERF-2 in a distinct pattern. NHERF-1 is strongly expressed in microvilli in association with NHE3, Npt2, and ezrin. Although NHERF-2 can be detected weakly in the microvilli, it is expressed predominantly at the base of the microvilli in the vesicle-rich domain. NHERF-2 appears to associate directly with ezrin and NHE3 but not Npt2. NHERF-1 is involved in the apical expression of Npt2 and the presence of other Npt2-binding proteins does not compensate totally for the absence of NHERF-1 in NHERF-1-null mice. Although NHERF-1 links NHE3 to the actin cytoskeleton through ezrin, the absence of NHERF-1 does not result in a generalized disruption of the architecture of the cell. Thus the mistargeting of Npt2 seen in NHERF-1-null mice likely represents a specific disruption of pathways mediated by NHERF-1 to achieve targeting of Npt2. These findings suggest that the organized subcellular distribution of the NHERF isoforms may play a role in the specific interactions mediating physiological control of transporter function.