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1.
Histol Histopathol ; 27(12): 1559-77, 2012 12.
Artigo em Inglês | MEDLINE | ID: mdl-23059887

RESUMO

Potassium depletion (K⁺-D) induces hypertrophy and hyperplasia of collecting duct cells, and potassium repletion (K⁺-R) induces regression of these changes. The purpose of this study was to examine the time courses of the changes in cellular composition, the origin of intercalated cells (ICs) and the mechanism responsible for these changes. SD rats received K⁺-depleted diets for 1, 7, or 14 days. After K⁺-D for 14 days some of the rats received normal diets for 1, 3, 5, or 7 days. In the inner stripe of the outer medulla, K⁺-D increased significantly the number and proportion of H⁺-ATPase-positive ICs, but decreased the proportion of H⁺-ATPase-negative principal cells (PCs). However, proliferation was limited to H⁺-ATPase-negative PCs. During K⁺-R, the cellular composition was recovered to control level. Apoptosis increased during K⁺-R and exclusively limited in H⁺-ATPase-negative PCs. Double immunolabeling with antibodies to PC and IC markers identified both cells negative or positive for all markers during both K⁺-D and K⁺-R. Electron microscopic observation showed that ultrastructure of AE1-positive some cells were similar to AE1-negative some cells during K⁺-R. LC3 protein expression increased significantly and autophagic vacuoles appeared particularly in PCs on days 14 of K⁺-D and in ICs on days 3 of K⁺-R. These results suggest that PCs and ICs may interconvert in response to changes in dietary K+ availability and that autophagic pathways may be involved in the interconversion.


Assuntos
Medula Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Potássio/metabolismo , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Aquaporina 2/metabolismo , Autofagia , Proliferação de Células , Homeostase , Hiperplasia , Hipertrofia , Hipopotassemia/metabolismo , Hipopotassemia/patologia , Medula Renal/patologia , Medula Renal/ultraestrutura , Túbulos Renais Coletores/patologia , Túbulos Renais Coletores/ultraestrutura , Masculino , Microscopia Imunoeletrônica , Deficiência de Potássio/metabolismo , Deficiência de Potássio/patologia , Potássio na Dieta/administração & dosagem , ATPases Translocadoras de Prótons/metabolismo , Ratos , Ratos Sprague-Dawley
2.
Am J Transplant ; 7(3): 550-9, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17173655

RESUMO

Research suggests that end-stage renal disease patients with elevated body mass index (BMI) have superior outcomes on dialysis. In contrast, low and high BMI patients represent the highest risk cohorts for kidney transplant recipients. The important question remains concerning how to manage transplant candidates given the potentially incommensurate impact of BMI by treatment modality. We conducted a retrospective analysis of waitlisted and transplanted patients in the United States from 1990 to 2003. We constructed Cox models to evaluate the effect of BMI on mortality of waitlisted candidates and identified risk factors for rapid weight change. We then assessed the impact of weight change during waitlisting on transplant outcomes. Decline in BMI on the waiting list was not protective for posttransplant mortality or graft loss across BMI strata. Substantial weight loss pretransplantation was associated with rapid gain posttransplantation. The highest risk for death was among listed patients with low BMI (13-20 kg/m(2), adjusted hazard ratio = 1.47, p < 0.01). Approximately one-third of candidates had a change in BMI category prior to transplantation. While observed declines in BMI may be volitional or markers of disease processes, there is no evidence that candidates have improved transplant outcomes attributable to weight loss. Prospective trials are needed to evaluate the efficacy of weight loss protocols for candidates of kidney transplantation.


Assuntos
Índice de Massa Corporal , Falência Renal Crônica/mortalidade , Transplante de Rim , Listas de Espera , Redução de Peso , Adolescente , Adulto , Idoso , Peso Corporal , Feminino , Humanos , Falência Renal Crônica/cirurgia , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Resultado do Tratamento
3.
Transfus Clin Biol ; 13(1-2): 159-63, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16564723

RESUMO

A novel family of proteins, the Mep/AMT/Rh glycoprotein family may mediate important roles in transmembrane ammonia transport in a wide variety of single-celled and multicellular organisms. Results from our laboratory have examined the expression of the non-erythroid proteins, Rh B Glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), in a wide variety of mammalian tissues. In the kidney, Rhbg and Rhcg are present in distal nephron sites responsible for ammonia secretion. In the mouse kidney, Rhbg immunoreactivity is exclusively basolateral and Rhcg immunoreactivity is exclusively apical, whereas in the rat kidney Rhcg exhibits both apical and basolateral expression. Chronic metabolic acidosis increases Rhcg expression in the outer and inner medulla of the rat kidney; these changes, at least in the outer medullary collecting duct, involve changes in total cellular protein expression in both principal and intercalated cell and changes in its subcellular localization. In the liver, Rhbg is present in the basolateral plasma membrane of the perivenous hepatocyte and Rhcg is present in bile duct epithelia. In the gastrointestinal tract, Rhbg and Rhcg exhibit cell-specific, axially heterogeneous, and polarized expression. These patterns of expression are consistent with Rhbg and Rhcg mediating important roles in mammalian ammonia biology. The lack of the effect of chronic metabolic acidosis on Rhbg expression raises the possibility that Rhbg may function either as ammonia sensing-protein or that it may mediate roles other than ammonia transport.


Assuntos
Amônia/metabolismo , Proteínas de Transporte de Cátions/biossíntese , Glicoproteínas/biossíntese , Hepatócitos/metabolismo , Túbulos Renais Coletores/metabolismo , Túbulos Renais Distais/metabolismo , Glicoproteínas de Membrana/biossíntese , Proteínas de Membrana Transportadoras/biossíntese , Acidose/metabolismo , Animais , Ductos Biliares/metabolismo , Proteínas de Transporte de Cátions/genética , Polaridade Celular , Células Epiteliais/metabolismo , Feminino , Trato Gastrointestinal/citologia , Trato Gastrointestinal/metabolismo , Regulação da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Especificidade de Órgãos , RNA Mensageiro/biossíntese , Ratos , Especificidade da Espécie , Frações Subcelulares/metabolismo
4.
Acta Physiol Scand ; 179(4): 331-8, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14656370

RESUMO

A family of ammonium transporter proteins was recently identified. Members of this family, Rh B Glycoprotein (RhBG) and Rh C Glycoprotein (RhCG) are expressed in the kidney and the liver, important tissues for ammonium metabolism. Immunohistochemical studies demonstrate basolateral RhBG immunoreactivity in the connecting segment (CNT) and collecting ducts, but not in the proximal tubule or the loop of Henle. Colocalization with thiazide sensitive cotransporter and carbonic anhydrase II confirms expression in the CNT, initial collecting tubule (ICT), and throughout the collecting duct. Colocalization with AE1 and pendrin demonstrates expression is greatest in A-type intercalated cells in the cortical collecting duct (CCD), outer medullary collecting duct (OMCD) and inner medullary collecting duct (IMCD), present in the CCD principal cell, and not detectable in either pendrin-positive CCD intercalated cells or in non-intercalated cells in the OMCD and IMCD. RhCG immunoreactivity has a similar axial distribution as RhBG. However, RhCG immunoreactivity is apical, and is detectable in all CCD and outer stripe of OMCD cells. The liver, a second organ involved in ammonia metabolism, also expresses both RhBG and RhCG. Basolateral RhBG immunoreactivity is present in the perivenous hepatocyte, but is not present in either the periportal or mid-zonal hepatocyte. Hepatic RhCG mRNA is expressed at lower levels than RhBG, and RhCG protein is detected in bile duct epithelium. These findings indicate that RhBG and RhCG are involved in at least two organs that transport ammonia, and that they are located in sites where they are likely to mediate important roles in ammonia transport.


Assuntos
Proteínas de Transporte/análise , Proteínas de Transporte de Cátions/análise , Glicoproteínas/análise , Rim/metabolismo , Fígado/metabolismo , Glicoproteínas de Membrana/análise , Compostos de Amônio Quaternário/metabolismo , Animais , Transporte Biológico , Humanos , Túbulos Renais Coletores/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , RNA Mensageiro/análise
5.
Am J Physiol Renal Physiol ; 278(2): F219-26, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10662726

RESUMO

Both acidosis and hypokalemia stimulate renal ammoniagenesis, and both regulate urinary proton and potassium excretion. We hypothesized that ammonia might play an important role in this processing by stimulating H(+)-K(+)-ATPase-mediated ion transport. Rabbit cortical collecting ducts (CCD) were studied using in vitro microperfusion, bicarbonate reabsorption was measured using microcalorimetry, and intracellular pH (pH(i)) was measured using the fluorescent, pH-sensitive dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Ammonia caused a concentration-dependent increase in net bicarbonate reabsorption that was inhibited by luminal addition of either of the H(+)-K(+)-ATPase inhibitors, Sch-28080 or ouabain. The stimulation of net bicarbonate reabsorption was not mediated through apical H(+)-ATPase, basolateral Na(+)-K(+)-ATPase, or luminal electronegativity. Although ammonia caused intracellular acidification, similar changes in pH(i) induced by inhibiting basolateral Na(+)/H(+) exchange did not alter net bicarbonate reabsorption. We conclude that ammonia regulates CCD proton and potassium transport, at least in part, by stimulating apical H(+)-K(+)-ATPase.


Assuntos
Amônia/farmacologia , Bicarbonatos/farmacocinética , Imidazóis/farmacologia , Túbulos Renais Coletores/efeitos dos fármacos , Inibidores da Bomba de Prótons , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Ouabaína/farmacologia , Coelhos
6.
Am J Physiol ; 276(4): F606-13, 1999 04.
Artigo em Inglês | MEDLINE | ID: mdl-10198421

RESUMO

The inner stripe of outer medullary collecting duct (OMCDis) is unique among collecting duct segments because both intercalated cells and principal cells secrete protons and reabsorb luminal bicarbonate. The current study characterized the mechanisms of OMCDis proton secretion. We used in vitro microperfusion, and we separately studied the principal cell and intercalated cell using differential uptake of the fluorescent, pH-sensitive dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Both the principal cell and intercalated cell secreted protons, as identified as Na+/H+ exchange-independent intracellular pH (pHi) recovery from an intracellular acid load. Two proton transport activities were identified in the principal cell; one was luminal potassium dependent and Sch-28080 sensitive and the other was luminal potassium independent and luminal bafilomycin A1 sensitive. Thus the OMCDis principal cell expresses both apical H+-K+-ATPase and H+-ATPase activity. Intercalated cell Na+/H+ exchange-independent pHi recovery was approximately twice that of the principal cell and was mediated by pharmacologically similar mechanisms. We conclude 1) the OMCDis principal cell may contribute to both luminal potassium reabsorption and urinary acidification, roles fundamentally different from those of the principal cell in the cortical collecting duct; and 2) the OMCDis intercalated cell proton transporters are functionally similar to those in the principal cell, raising the possibility that an H+-K+-ATPase similar to the one present in the principal cell may contribute to intercalated cell proton secretion.


Assuntos
Túbulos Renais Coletores/metabolismo , Macrolídeos , Animais , Antibacterianos/farmacologia , Membrana Celular/metabolismo , Inibidores Enzimáticos/farmacologia , ATPase Trocadora de Hidrogênio-Potássio/fisiologia , Concentração de Íons de Hidrogênio , Imidazóis/farmacologia , Medula Renal , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Inibidores da Bomba de Prótons , Bombas de Próton/fisiologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/fisiologia , Prótons , Coelhos
7.
Am J Physiol ; 276(2): F237-45, 1999 02.
Artigo em Inglês | MEDLINE | ID: mdl-9950954

RESUMO

In the present study, we demonstrate that the rabbit cortical collecting duct cell line RCCT-28A possesses three distinct H-K-ATPase catalytic subunits (HKalpha). Intracellular measurements of RCCT-28A cells using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) indicated that the mechanism accounting for recovery from an acid load exhibited both K+ dependence and sensitivity to Sch-28080 characteristic of H-K-ATPases. Recovery rates were 0.022 +/- 0.005 pH units/min in the presence of K+, 0.004 +/- 0.002 in the absence of K+, and 0.002 +/- 0.002 in the presence of Sch-28080. The mRNAs encoding the HKalpha1 subunit and the H-K-ATPase beta-subunit (HKbeta) were detected by RT-PCR. In addition, two HKalpha2 species were found by RT-PCR and 5' rapid amplification of cDNA ends (5'-RACE) in the rabbit renal cortex. One was homologous to HKalpha2 cDNAs generated from other species, and the second was novel. The latter, referred to as HKalpha2c, encoded an apparent 61-residue amino-terminal extension that bore no homology to reported sequences. Antipeptide antibodies were designed on the basis of this extension, and these antibodies recognized a protein of the appropriate mass in both rabbit renal tissue samples and RCCT-28A cells. Such findings constitute very strong evidence for expression of the HKalpha2c subunit in vivo. The results suggest that the rabbit kidney and RCCT-28A cells express at least three distinct H-K-ATPases.


Assuntos
ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Túbulos Renais Coletores/enzimologia , Animais , Linhagem Celular , Colo/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/genética , Concentração de Íons de Hidrogênio , Isoenzimas/genética , Córtex Renal/metabolismo , Túbulos Renais Coletores/citologia , Potássio/farmacologia , RNA Mensageiro/metabolismo , Coelhos
8.
Am J Physiol ; 274(6): F1086-94, 1998 06.
Artigo em Inglês | MEDLINE | ID: mdl-9841500

RESUMO

The cortical collecting duct (CCD) B cell possesses an apical anion exchanger dissimilar to AE1, AE2, and AE3. The purpose of these studies was to characterize this transporter more fully by examining its regulation by CO2 and HCO3. We measured intracellular pH (pHi) in single intercalated cells of in vitro microperfused CCD using the fluorescent, pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In the absence of extracellular CO2/HCO3, luminal Cl removal caused reversible intracellular alkalinization, identifying this transporter as a Cl/base exchanger able to transport bases other than HCO3. Adding extracellular CO2/HCO3 decreased B cell pHi while simultaneously increasing Cl/base exchange activity. Since intracellular acidification inhibits AE1, AE2, and AE3, we examined mechanisms other than pHi by which the stimulation occurred. These studies showed that B cell apical anion exchange activity was CO2 stimulated and carbonic anhydrase dependent. Moreover, the stimulation was independent of luminal bicarbonate, luminal pH or pHi, and changes in buffer capacity. We conclude that the B cell possesses an apical Cl/base exchanger whose activity is regulated by CO2-stimulated, carbonic anhydrase-dependent cytoplasmic HCO3 formation.


Assuntos
Antiporters/metabolismo , Bicarbonatos/metabolismo , Dióxido de Carbono/metabolismo , Túbulos Renais Coletores/metabolismo , Animais , Inibidores da Anidrase Carbônica/farmacologia , Antiportadores de Cloreto-Bicarbonato , Feminino , Concentração de Íons de Hidrogênio , Coelhos
10.
Am J Physiol ; 273(3 Pt 2): F340-7, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9321906

RESUMO

The A cell may possess multiple H+ transporters, including H(+)-adenosinetriphosphatase (H(+)-ATPase) and H(+)-K(+)-ATPase. The current study examines the relative roles of proton transporters in the A cell by observing their contribution to both basal intracellular pH (pHi) regulation and pHi recovery from an intracellular acid load. CCD were studied using in vitro microperfusion, and pHi was measured in the individual A cell using the fluorescent, pH-sensitive dye, 2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein (BCECF). Inhibiting H(+)-ATPase with luminal bafilomycin A1 decreased basal pHi, whereas inhibiting apical H(+)-K(+)-ATPase with either luminal Sch-28080 or luminal potassium removal did not. The predominant mechanism of pHi, recovery from an intracellular acid load was peritubular sodium dependent and peritubular ethylisopropylamiloride (EIPA) sensitive, identifying basolateral Na+/H+ exchange activity. In the absence of peritubular sodium, pHi recovery was inhibited by luminal bafilomycin A1 but not by luminal Sch-28080 addition or by luminal potassium removal. However, when Na+/H+ exchange was inhibited with EIPA, both bafilomycin A1 sensitive and potassium dependent, Sch-28080-sensitive components of pHi recovery were present. Quantitatively, the rate of H(+)-ATPase proton secretion was greater than the rate of H(+)-K(+)-ATPase proton secretion. We conclude that basolateral Na+/H+ exchange is the predominant mechanism of A cell pHi recovery from an intracellular acid load. An apical H(+)-ATPase is the primary apical transporter contributing to A cell pHi regulation. An apical H(+)-K(+)-ATPase, while present, plays a more limited role under the conditions tested.


Assuntos
Concentração de Íons de Hidrogênio , Córtex Renal/fisiologia , Túbulos Renais Coletores/fisiologia , Macrolídeos , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Homeostase , Imidazóis/farmacologia , Técnicas In Vitro , Líquido Intracelular/fisiologia , Córtex Renal/citologia , Túbulos Renais Coletores/citologia , Cinética , Perfusão , Inibidores da Bomba de Prótons , ATPases Translocadoras de Prótons/antagonistas & inibidores , Coelhos , Sódio/metabolismo
13.
Am J Physiol ; 270(3 Pt 2): F518-30, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8780256

RESUMO

The role of H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) in the cortical collecting duct (CCD) B-type intercalated cell (B cell) is unclear. This study examined whether H(+)-K(+)-ATPase contributes to B cell intracellular pH (pHi) regulation and, if so, whether it is present at the apical or basolateral membrane. B cell Na(+)-independent pHi recovery from an acid load was only partially inhibited by peritubular N-ethylmaleimide (NEM). Complete inhibition required combining peritubular NEM either with luminal Sch-28080 or with luminal K+ removal. In contrast, neither peritubular Sch-28080 nor peritubular K+ removal altered pHi regulation. Tomato lectin, which binds to the gastric H(+)-K(+)-ATPase beta-subunit, labeled the B cell apical membrane. We conclude that the rabbit CCD B cell possesses an apical H(+)-K(+)-ATPase that plays an important role in pHi recovery from an in vitro acid load.


Assuntos
ATPase Trocadora de Hidrogênio-Potássio/análise , Córtex Renal/enzimologia , Túbulos Renais Coletores/enzimologia , Animais , Membrana Celular/enzimologia , Feminino , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Concentração de Íons de Hidrogênio , Coelhos
15.
Am J Physiol ; 269(5 Pt 2): F730-8, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7503240

RESUMO

Angiotensin II (ANG II) regulates whole kidney ion transport, yet its effects in the collecting duct are unknown. The purpose of these studies was to determine whether ANG II regulates luminal alkalinization and acidification in the rabbit cortical collecting duct (CCD). The rate of luminal alkalinization or acidification was measured as the rate of change of luminal fluid pH under stop-flow conditions using in vitro microperfused CCD segments. Outer CCD alkalinized the luminal fluid, consistent with net HCO3- secretion. Addition of ANG II, 10(-7) M, to the peritubular solution for 30 min significantly stimulated luminal alkalinization. The stimulatory effect of ANG II was not due to time-dependent effects and was blocked by peritubular addition of the ANG II type 1 (AT1) receptor antagonist, losartan, at 10(-6) M. Losartan, 10(-6) M, when added to the peritubular solution, did not alter the rate of luminal alkalinization independent of ANG II. In contrast, peritubular ANG II, 10(-7) M, did not alter inner CCD luminal acidification. Addition of ANG II to the peritubular solution at the lower concentration of 10(-10) M did not alter the rates of luminal alkalinization and acidification in the outer and inner CCD, respectively. Peritubular ANG II, 10(-7) M, but not vehicle, stimulated B cell apical HCO3- secretion occurring in response to peritubular Cl- removal. These studies demonstrate that ANG II acts through a basolateral AT1 receptor to stimulate outer CCD luminal alkalinization via, at least in part, B cell stimulation.


Assuntos
Ácidos/metabolismo , Álcalis/metabolismo , Angiotensina II/farmacologia , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Angiotensina I/metabolismo , Antagonistas de Receptores de Angiotensina , Animais , Bicarbonatos/metabolismo , Compostos de Bifenilo/farmacologia , Feminino , Concentração de Íons de Hidrogênio , Imidazóis/farmacologia , Técnicas In Vitro , Losartan , Concentração Osmolar , Perfusão , Coelhos , Tetrazóis/farmacologia
16.
Am J Physiol ; 267(6 Pt 2): F952-64, 1994 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7810703

RESUMO

At least two cortical collecting duct (CCD) intercalated cell populations mediate HCO3- secretion and reabsorption. The present study examined the membrane location of intercalated cell Cl-/base exchange activity and the axial distribution of CCD intercalated cells. CCD were studied using in vitro microperfusion in CO2/HCO3(-)-containing solutions; intracellular pH was measured using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The A-type intercalated cell (A cell) and B-type intercalated cell (B cell) were identified functionally by the absence and presence of apical Cl-/HCO3- exchange activity, respectively. When a 0 mM Cl-, 0 mM HCO3- luminal solution was used, removal of Cl- from the peritubular solution caused intracellular alkalinization in all B cells. The alkalinization required neither extracellular Na+ nor changes in membrane potential. Peritubular 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (10(-4) M) inhibited A cell but not B cell basolateral Cl-/base exchange activity. In comparison to studies performed with a 0 mM Cl- 0 mM HCO3- luminal solution, the use of a 0 mM Cl-, 25 mM HCO3- luminal solution inhibited both the identification and the magnitude of B cell basolateral Cl-/base exchange activity. When CCD from the inner and outer cortex were separately studied, only 7% of outer CCD intercalated cells were A cells, whereas 93% were B cells. In contrast, in the inner CCD, 58% of intercalated cells were A cells and 42% were B cells. Under stop-flow conditions, outer CCD alkalinized the luminal fluid, whereas inner CCD acidified the luminal fluid. These results indicate that all CCD intercalated cells possess basolateral Cl-/base exchange activity; however, A cell and B cell basolateral Cl-/base exchange activity differs, at least in terms of sensitivity to DIDS. Furthermore, there is axial heterogeneity in both intercalated cell type and function.


Assuntos
Antiporters/metabolismo , Córtex Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Bicarbonatos/metabolismo , Antiportadores de Cloreto-Bicarbonato , Cloretos/metabolismo , Endocitose , Fluoresceínas/metabolismo , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Potenciais da Membrana , Coelhos , Sódio/farmacologia
17.
Am J Physiol ; 266(3 Pt 2): F466-76, 1994 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8160796

RESUMO

The inner medullary collecting duct (IMCD) is the final portion of the mammalian renal tubule that is able to significantly regulate systemic acid-base balance. Although the H+ transporters of this segment are relatively well studied, little is known regarding the mechanisms of HCO3- transport. The mechanisms of HCO3- transport in primary cultures of rabbit IMCD were studied using the pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, in CO2/HCO3(-)-containing solutions at 37 degrees C. Removal of Cl- from the extracellular solution caused reversible intracellular alkalinization, demonstrating the presence of Cl-/HCO3- exchange. Alkalinization with Cl- removal was independent of changes in membrane potential, did not require the presence of extracellular Na+, and was inhibited by the disulfonic stilbene, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 10(-4) M). Half-maximal intracellular pH (pHi) recovery with readdition of Cl- to the extracellular solution occurred at a Cl- concentration of 37.4 +/- 5.7 mM. When rabbit IMCD were cultured on permeable support membranes, Cl-/HCO3- exchange activity was found only on the basolateral membrane. However, there was no evidence of band 3 protein immunoreactivity. In contrast, no evidence for Na(+)-(HCO3-)n > 1 cotransport activity was found. Depolarization of IMCD cells by acute increases in extracellular K+ did not alter pHi, nor was Na(+)-dependent, 5-(N-ethyl-N-isopropyl)amiloride-insensitive pHi recovery from an acid load inhibited by DIDS (10(-4) M). Finally, recovery from intracellular alkalosis induced by incubation in 0 mM Cl-, 50 mM HCO3- extracellular solution required Cl- and was independent of Na+. These studies indicate that the major mechanism of HCO3- transport in primary cultures of the rabbit IMCD is via a band 3 protein-negative, Na(+)-independent, basolateral, Cl-/HCO3- exchanger.


Assuntos
Bicarbonatos/metabolismo , Túbulos Renais Coletores/metabolismo , Álcalis/metabolismo , Animais , Proteína 1 de Troca de Ânion do Eritrócito/imunologia , Anticorpos/imunologia , Antiporters/metabolismo , Transporte Biológico , Células Cultivadas , Antiportadores de Cloreto-Bicarbonato , Cloretos/metabolismo , Membranas Intracelulares/metabolismo , Medula Renal , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/fisiologia , Potenciais da Membrana , Coelhos , Sódio/fisiologia , Estilbenos/farmacologia
18.
Am J Physiol ; 265(3 Pt 2): F406-15, 1993 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8214100

RESUMO

The inner stripe of the outer medullary collecting duct (OMCDis) is a major site of HCO3- reabsorption and urinary acidification. Whether this nephron segment consists of a single or multiple cell types remains unclear. Apical incubation of rabbit OMCDis via luminal perfusion with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester resulted in heterogeneous fluorescence, suggesting two cell types. This heterogeneity was not prevented by inhibition of either carbonic anhydrase or organic anion transport. Subsequent studies were directed at characterizing the major intracellular pH (pHi) regulatory transporters in these two cell populations. Both cell populations demonstrated similar rates of Na+/H+ exchange, as assessed by peritubular Na(+)-dependent, amiloride-sensitive pHi recovery from an intracellular acid load. In contrast, Na(+)-independent, HCO3(-)-independent pHi recovery from an acid load was present in both cell populations but had two to three times greater activity in a minority cell population. In vivo deoxycorticosterone acetate administration increases this rate in both populations but to a greater extent in the minority cell population. In CO2/HCO3(-)-containing solutions, Cl- removal from the peritubular solution caused 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive alkalinization of all cells. Again, the magnitude and rate of alkalinization were significantly greater in the minority cell population. These studies demonstrate that the OMCDis consists of qualitatively similar cells in different states of functional activity. Although they are similar in most characteristics, a minority of cells more actively secrete H+ (independent of Na+) and reabsorb HCO3-.


Assuntos
Membranas Intracelulares/metabolismo , Túbulos Renais Coletores/fisiologia , Animais , Antiporters/metabolismo , Soluções Tampão , Antiportadores de Cloreto-Bicarbonato , Desoxicorticosterona/farmacologia , Fluoresceínas/farmacologia , Concentração de Íons de Hidrogênio , Processamento de Imagem Assistida por Computador , Técnicas In Vitro , Medula Renal , Túbulos Renais Coletores/citologia , Coelhos , Sódio/farmacologia , Trocadores de Sódio-Hidrogênio/metabolismo , Televisão
20.
J Clin Invest ; 87(5): 1553-8, 1991 May.
Artigo em Inglês | MEDLINE | ID: mdl-2022727

RESUMO

Cl-/HCO3- exchange is present in all three cell types of the rabbit cortical collecting tubule, yet may mediate a different function in each cell type. The purpose of this study was to characterize further the location, function, and regulation of Cl-/HCO3- exchange in two cell types using measurements of intracellular pH (pHi). In the principal cell there was no evidence for apical Cl-/HCO3- exchange, including no change in pHi with increases in luminal HCO3-. The principal cell possesses a basolateral Cl-/HCO3- exchanger that is inactive normally but stimulated by intracellular alkalosis. Decreased PCO2 results in increased pHi associated with activation of Cl-/HCO3- exchange and partial recovery of pHi. In contrast, the beta-intercalated cell possesses an apical Cl-/HCO3- exchanger and alkalinizes with increases in luminal HCO3-. Also in contrast to the principal cell, the beta-intercalated cell apical Cl-/HCO3- exchanger does not appear to be involved in pHi regulation and may be specifically modified for transcellular HCO3- transport. In conclusion, the separate Cl-/HCO3- exchangers in the principal cell and the beta-intercalated cell not only have opposite polarity but are regulated differently.


Assuntos
Bicarbonatos/metabolismo , Cloretos/metabolismo , Córtex Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Animais , Transporte Biológico , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Coelhos
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