Signaling pathways involved in the rapid biphasic effect of aldosterone on Na+/H+ exchanger in rat proximal tubule cells.

The receptors and signaling pathways for nongenomic effects of aldosterone (Aldo) on the proximal Na+/H+ exchanger are still unknown; therefore, the aim of this study was to investigate the mineralocorticoid receptor (MR) and/or glucocorticoid receptor (GR) participation in rapid Aldo effects on NHE1 (basolateral Na+/H+ exchanger isoform) and cytosolic calcium concentration ([Ca2+]i). In addition, phospholipase C (PLC), protein kinase C (PKC), and mitogen-activated protein kinase kinase (MEK) involvement in signaling pathways of such effects was evaluated, using immortalized proximal tubule cells of rat (IRPTC) as an experimental model. MR and GR expression was investigated using reverse transcription polymerase chain reaction and immunoblotting. The intracellular pH recovery rate (after acid loading) and [Ca2+]i were determined by the probes BCECF-AM and FURA 2-AM, respectively. Aldo (10-12 M) promoted a moderate increase in [Ca2+]i and stimulation of NHE1, whereas Aldo (10-6 M) greatly increased the [Ca2+]i, but inhibited the NHE1. BAPTA-AM (a calcium chelator), GR antagonism and inhibition of PLC, PKC and MEK pathway abolished the biphasic and dose-dependent effect of Aldo on NHE1 and decreased the [Ca2+]i; whereas MR do not appear to participate in this rapid signaling in IRPTC cells. The reduction of GR content, by gene silencing, abolished the Aldo effect on NHE1, in low concentration, confirming the importance of this receptor in the rapid modulation of proximal sodium and hydrogen transports.

The Role of ß-Adrenergic Overstimulation in the Early Stages of Renal Injury.

BACKGROUND/AIMS: To assess the possible contribution of the ß-adrenergic overstimulation in early stages of renal injury, the present study evaluated, in rats, the effects of the ß-adrenoceptor agonist isoproterenol (ISO) on renal function and morphology, as well as the renal mRNA and protein expression of the NADPH oxidase isoform 4 (Nox 4) and subunit p22phox, endoplasmic reticulum (ER) stress, pro-inflammatory, pro-apoptotic and renin-angiotensin system (RAS) components. METHODS: Wistar rats received ISO (0.3 mg.kg-1.day-1 s.c.) or vehicle (control) for eight days. At the end of the treatment, food and water intake, urine output and body weight gain were evaluated and renal function studies were performed. Renal tissue was used for the morphological, quantitative PCR and immunohistochemical studies. RESULTS: ISO did not change metabolic parameters or urine output. However it induced a decrease in renal blood flow and an increase in the filtration fraction. These changes were accompanied by increased cortical mRNA and protein expression for the renal oxidative stress components including Nox 4 and p22phox; ER stress, pro-inflamatory, pro-apoptotic as well as RAS components. ISO also induced a significant increase in medullar renin protein expression. CONCLUSION: These findings support relevant information regarding the contribution of specific ß-adrenergic hyperactivity in early stage of renal injury, indicating the reactive oxygen species, ER stress and intrarenal RAS as important factors in this process.

The effects of angiotensin-(1-7) on the exchanger NHE3 and on [Ca2+]i in the proximal tubules of spontaneously hypertensive rats.

The acute effects of angiotensin-1-7 [ANG-(1-7)] on the reabsorptive bicarbonate flow (J[Formula: see text]) were evaluated using stationary microperfusion in vivo in the proximal tubules of spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto (WKY) rats, using a microelectrode sensitive to H+ In WKY rats, the control J[Formula: see text] was 2.40 ± 0.10 nmol·cm-2·s-1 (n = 120); losartan (10-7 M) or A779 (10-6 M, a specific Mas antagonist), alone or in combination with losartan, decreased the J[Formula: see text] ANG-(1-7) had biphasic effects on J[Formula: see text]: at 10-9 M, it inhibited, and at 10-6, it stimulated the flow. S3226 [10-6 M, a specific Na+-H+ exchanger 3 (NHE3) antagonist] decreased J[Formula: see text] and changed the stimulatory effect of ANG-(1-7) to an inhibitory one but did not alter the inhibitory action of ANG-(1-7). In SHR, the control J[Formula: see text] was 2.04 ± 0.13 nmol·cm-2·s-1 (n = 56), and A779 and/or losartan reduced the flow. ANG-(1-7) at 10-9 M increased J[Formula: see text], and ANG-(1-7) at 10-6 M reduced it. The effects of A779, losartan, and S3226 on the J[Formula: see text] were similar to those found in WKY rats, which indicated that in SHR, the ANG-(1-7) action on the NHE3 was via Mas and ANG II type 1. The cytosolic calcium in the WKY or SHR rats was ~100 nM and was increased by ANG-(1-7) at 10-9 or 10-6 M. In hypertensive animals, a high plasma level of ANG-(1-7) inhibited NHE3 in the proximal tubule, which mitigated the hypertension caused by the high plasma level of ANG II.

Dose-dependent effects of angiotensin-(1-7) on the NHE3 exchanger and [Ca(2+)](i) in in vivo proximal tubules.

The acute direct action of angiotensin-(1-7) [ANG-(1-7)] on bicarbonate reabsorption (JHCO(3)(-)) was evaluated by stationary microperfusions on in vivo middle proximal tubules in rats using H ion-sensitive microelectrodes. The control JHCO(3)(-) is 2.82 ± 0.078 nmol·cm(-2)·s(-1) (50). ANG-(1-7) (10(-12) or 10(-9) M) in luminally perfused tubules decreases JHCO(3)(-) (36 or 60%, respectively), but ANG-(1-7) (10(-6) M) increases it (80%). A779 increases JHCO(3)(-) (30%) and prevents both the inhibitory and the stimulatory effects of ANG-(1-7) on it. S3226 decreases JHCO(3)(-) (45%) and changes the stimulatory effect of ANG-(1-7) to an inhibitory effect (30%) but does not affect the inhibitory effect of ANG-(1-7). Our results indicate that in the basal condition endogenous ANG-(1-7) inhibits JHCO(3)(-) and that the biphasic dose-dependent effect of ANG-(1-7) on JHCO(3)(-) is mediated by the Mas receptors via the Na(+)/H(+) exchanger 3 (NHE3). The control value of intracellular Ca(2+) concentration ([Ca(2+)](i)), as monitored using fura-2 AM, is 101 ± 2 nM (6), and ANG-(1-7) (10(-12), 10(-9), or 10(-6)M) transiently (3 min) increases it (by 151, 102, or 52%, respectively). A779 increases the [Ca(2+)](i) (25%) but impairs the stimulatory effect of all doses of ANG-(1-7) on it. The use of BAPTA or thapsigargin suggests a correlation between the ANG-(1-7) dose-dependent effects on [Ca(2+)](i) and JHCO(3)(-). Therefore, the interaction of the opposing dose-dependent effects of ANG II and ANG-(1-7) on [Ca(2+)](i) and JHCO(3)(-) may represent an physiological regulatory mechanism of extracellular volume and/or pH changes. However, whether [Ca(2+)](i) modification is an important direct mechanism for NHE3 activation by these peptides or is a side effect of other signaling pathways will require additional studies.

Regulation of Na+/H+ exchanger isoform 1 (NHE1) by calmodulin-binding sites: role of angiotensin II.

We examined the effect of Angiotensin II (Ang II) on the interaction between the Ca(2+)/CaM complex and hNHE1. Considering that calmodulin binds to NHE1 at two sites (A and B), amino acids at both sites were modified and two mutants were constructed: SA(1K3R/4E) and SB(1K3R/4E). Wild type and mutants were transfected into PS120 cells and their activity was examined by H(+) flux (J(H+)). The basal J(H+) of wild type was 4.71 ± 0.57 (mM/min), and it was similar in both mutants. However, the mutations partially impaired the binding of CaM to hNHE1. Ang II (10(-12) and 10(-9) M) increased the J(H+) in wild type and SB. Ang II (10(-6) M) increased this parameter only in SA. Ang II (10(-9) M) maintained the expression of calmodulin in wild type or mutants, and Ang II (10(-6) M) decreased it in wild type or SA, but not in SB. Dimethyl-Bapta-AM (10(-7) M), a calcium chelator, suppressed the effect of Ang II (10(-9) M) in wild type. With Ang II (10(-6) M), Bapta failed to affect wild type or SA, but it increased the J(H+) in SB. W13 or calmidazolium chloride (10(-5) M), two distinct calmodulin inhibitors, decreased the effect of Ang II (10(-9) M) in wild type or SB. With Ang II (10(-6) M), W13 or calmidazolium chloride decreased the J(H+) in wild type or SA and increased it in SB. Thus, with Ang II (10(-12) and 10(-9) M), site A seems to be responsible for the stimulation of hNHE1 and with Ang II (10(-6) M), site B is important to maintain its basal activity.

Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule.

The direct action of aldosterone (10(-12) M) on net bicarbonate reabsorption (J(HCO(3)(-))) was evaluated by stationary microperfusion of an in vivo middle proximal tubule (S2) of rat kidney, using H ion-sensitive microelectrodes. Aldosterone in luminally perfused tubules caused a significant increase in J(HCO(3)(-)) from a mean control value of 2.84 +/- 0.08 [49/19 (n degrees of measurements/n degrees of tubules)] to 4.20 +/- 0.15 nmol.cm(-2).s(-1) (58/10). Aldosterone perfused into peritubular capillaries also increased J(HCO(3)(-)), compared with basal levels during intact capillary perfusion with blood. In addition, in isolated perfused tubules aldosterone causes a transient increase of cytosolic free calcium ([Ca(2+)](i)), monitored fluorometrically. In the presence of ethanol (in similar concentration used to prepare the hormonal solution), spironolactone (10(-6) M, a mineralocorticoid receptor antagonist), actinomycin D (10(-6) M, an inhibitor of gene transcription), or cycloheximide (40 mM, an inhibitor of protein synthesis), the J(HCO(3)(-)) and the [Ca(2+)](i) were not different from the control value; these drugs also did not prevent the stimulatory effect of aldosterone on J(HCO(3)(-)) and on [Ca(2+)](i). However, in the presence of RU 486 alone [10(-6) M, a classic glucocorticoid receptor (GR) antagonist], a significant decrease on J(HCO(3)(-)) and on [Ca(2+)](i) was observed; this antagonist also inhibited the stimulatory effect of aldosterone on J(HCO(3)(-)) and on [Ca(2+)](i). These studies indicate that luminal or peritubular aldosterone (10(-12) M) has a direct nongenomic stimulatory effect on J(HCO(3)(-)) and on [Ca(2+)](i) in proximal tubule and that probably GR participates in this process. The data also indicate that endogenous aldosterone stimulates J(HCO(3)(-)) in middle proximal tubule.

The effect of angiotensin II on intracellular pH is mediated by AT(1) receptor translocation.

The effect of ANG II on intracellular pH (pH(i)) recovery rate and AT(1) receptor translocation was investigated in transfected MDCK cells. The pH(i) recovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin II receptor isoform 1 (hAT(1)) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pH(i) recovery rate of 0.219 +/- 0.017 pH U/min (n = 11). This value was similar to nontransfected cells [0.211 +/- 0.009 pH U/min (n = 12)]. Both values were significantly increased with ANG II (10(-9) M) but not with ANG II (10(-6) M). Losartan (10(-7) M) and dimethyl-BAPTA-AM (10(-7) M) decreased significantly the stimulatory effect of ANG II (10(-9) M) and induced an increase in Na(+)/H(+) exchanger 1 (NHE-1) activity with ANG II (10(-6) M). Immunofluorescence studies indicated that in control situation, the hAT(1) receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II (10(-9) M) and internalized with ANG II (10(-6) M). Losartan (10(-7) M) induced hAT(1) translocation to plasma membrane in all studied groups. Dimethyl-BAPTA-AM (10(-7) M) did not change the effect of ANG II (10(-9) M) on the hAT(1) receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II (10(-6) M). With ionomycin (10(-6) M), the receptor was accumulated in cytosol. The results indicate that, in MDCK cells, the effect of ANG II on NHE-1 activity is associated with ligand binding to AT(1) receptor and intracellular signaling events related to AT(1) translocation.

Action of ANG II and ANP on colon epithelial cells.

The interaction of angiotensin II (ANG II) and atrial natriuretic peptide (ANP) on intracellular pH (pH(i)) and calcium ([Ca2+](i)) was investigated in T84 cells (a permanent cell line derived from human colon epithelium) using the fluorescent stains BCECF/AM and Fluo 4/AM, respectively. pH(i) recovery rate mediated by the Na(+)/H+ exchanger (NHE) was examined following an NH4Cl pulse. Under control conditions pH(i) recovered at 0.114+/-0.005 pH units/min (n=35). ANG II (10(-12) or 10(-9) M) increased this value, whilst ANG II (10(-7) M) decreased it. These effects of ANG II were impaired by simultaneous addition of 1 microM or 25 microM HOE-694, indicating that the stimulatory and inhibitory effects of ANG II on pH(i) recovery are mediated in part via the NHE1 and NHE2 isoforms. ANG II increased [Ca2+]i concentration-dependently. ANP (10(-6) M) or dimethyl-BAPTA/AM (50 microM) blocked the effects of ANG II on [Ca2+]i and on the rate of pH(i) recovery. Thapsigargin (10(-5) M) enhanced the effect of ANG II on [Ca2+]i and reversed its stimulatory effect on the rate of pH(i) recovery to an inhibitory one. External Ca(2+)-free solution did not affect the effects of ANG II on these parameters. These data suggest that the [Ca2+]i increase induced by ANG II is dependent on intracellular calcium stores. They are compatible with the demonstration of two sites on the C-terminal of the Na(+)/H+ exchanger, one stimulating Na(+)/H+ activity by increases of [Ca2+]i in the lower range (at 10(-12) or 10(-9) M ANG II) and the other inhibiting this activity at high [Ca2+]i levels (at 10(-7) M ANG II). ANP or dimethyl-BAPTA/AM, by impairing the pathway mediating the increase in [Ca2+]i, block both the stimulatory and inhibitory effects of ANG II.

Signaling path of the action of AVP on distal K+ secretion.

BACKGROUND: Previous studies from our laboratory have shown that luminal perfusion with arginine vasopressin (AVP) stimulates distal tubule secretory potassium flux (JK) via V1 receptors (Am J Physiol 278:F809-F816, 2000). In the present work, we investigate the cell signaling mechanism of this process. METHODS: In vivo stationary microperfusion was performed in rat cortical distal tubules and luminal K+ was measured using double K+ resin/reference microelectrodes. RESULTS: In control conditions, JK was 0.71 +/- 0.05 nmol.cm(-2).second(-1); this process was inhibited (14%) by 10(-5) mol/L 8-bromo-cyclic adenosine monophosphate (cAMP), and increased by 35% with 10(-8) mol/L phorbol ester [phorbol12-myristate 13-acetate (PMA), which activates protein kinase C (PKC)]. During luminal perfusion with 10(-11) mol/L AVP, JK increased to 0.88 +/- 0.08 nmol.cm(-2).seconds(-1). In the presence of 10(-11) mol/L AVP, JK was not affected by 10(-4) mol/L H89, a blocker of protein kinase A (PKA), but was inhibited (45%) by 10(-5) mol/L staurosporine, an inhibitor of PKC, and by 41% during perfusion with 5 x 10(-5) mol/L of the cell Ca2+ chelator bis (2-aminophenoxy) ethane-tetraacetic acid (BAPTA). In order to study the role of Ca(2+)-dependent K channels in the luminal hormonal action, the tubules were perfused with 5 mmol/L tetraethylammonium chloride (TEA) or 10(-7) mol/L iberiotoxin, in the presence of AVP, and JK was significantly reduced by both agents. Iberiotoxin reduced AVP-stimulated JK by 36.4%, and AVP-independent JK (after blocking V1 receptors) by only 16%. CONCLUSION: The results suggest that the luminal V1-receptor effect of AVP on JK was mediated by the phospholipase C (PLC)/Ca2+/PKC signaling path and not byadenylate cyclase/cAMP/PKA, therefore probably acting on maxi-potassium channels.

Arginine vasopressin stimulates H+-ATPase in MDCK cells via V1 (cell Ca2+) and V2 (cAMP) receptors.

The effect of arginine vasopressin (AVP) and/or atrial natriuretic peptide (ANP) on the regulation of intracellular pH (pHi) via H+-ATPase and of cytosolic calcium ([Ca2+]i) was investigated in Madin-Darby canine kidney (MDCK) cells by the fluorescent probes BCECF-AM and fluo-4-AM, respectively. The pHi recovery rate was examined after intracellular acidification following an NH4Cl pulse, in the presence of zero Na+ plus Schering 28080 (a specific inhibitor of H+-K+-ATPase). AVP (10-12-10-6 M) increased the rate of pHi recovery and [Ca2+]i in a dose-dependent manner. V1- or V2-receptor antagonists impaired the effect of AVP on both processes, and DDAVP (10-12-10-6 M; a V2-selective agonist) caused a dose-dependent stimulation of them. [Ca2+]i or cAMP (as increased by 10-5 M thapsigargin or 8-BrcAMP, respectively) alone had no effect on H+-ATPase, but their synergic action was necessary to stimulate H+-ATPase. In agreement with these findings, ANP (10-6 M) or dimethyl-BAPTA-AM (5 x 10-5 M), impairing the increase of [Ca2+]i in response to AVP, blocks the stimulatory effect of AVP on H+-ATPase.

Atrial natriuretic peptide impairs the stimulatory effect of angiotensin II on H+-ATPase.

BACKGROUND: Angiotensin II (Ang II) action on H+-ATPase is not clearly defined, and may vary with renal tubule segment and hormonal doses being studied. Since an increase of cytosolic calcium ([Ca2+]i) can stimulate acid vesicle movement and exocytotic insertion of proton pumps, and it has been shown that Ang II increases [Ca2+]i while atrial natriuretic peptide (ANP) reduces it, there may be some interaction between Ang II and ANP in the regulation of intracellular pH (pHi) mediated by H+-ATPase. METHODS: The effects of Ang II and/or ANP on the regulation of pHi via H+-ATPase and of [Ca2+]i was investigated in Madin-Darby canine kidney cells (MDCK) by the fluorescent probes BCECF-AM and Fluo-4/AM, respectively. The pHi recovery rate was examined following the intracellular acidification after an NH4Cl pulse, in presence of zero Na+ plus Schering 28080, which is a specific inhibitor of H+/K+-ATPase. RESULTS: Ang II (10-12, 10-9 or 10-7 mol/L) increased the rate of pHi recovery and [Ca2+]i in a dose-dependent manner. ANP (10-6 mol/L) or dimethyl-BAPTA/AM (5 x 10-5 mol/L, an intracellular calcium chelator) did not affect the pHi recovery but decreased [Ca2+]i and blocked the stimulatory effect of Ang II on the pHi recovery. CONCLUSIONS: The results suggest that the increase of [Ca2+]i regulates the dose-dependent stimulatory effect of Ang II on H+-ATPase. ANP or dimethyl-BAPTA/AM, by impairing the path causing the increase in [Ca2+]i, blocks this stimulatory effect of Ang II.

Distal tubule bicarbonate transport.

The superficial cortical distal tubule, accessible to in vivo micropuncture and microperfusion, has been the site of a considerable number of investigations. An important fraction of renal tubule bicarbonate reabsorption, about 8 to 9% of the filtered load of this ion in the rat, takes place in this segment. The present review addresses several aspects of bicarbonate transport by distal tubule. It includes the overall magnitude of bicarbonate reabsorption, as well as the most important methods used to measure this process. The acid-base transporters responsible for transmembrane and transepithelial bicarbonate transport are also discussed. This analysis is followed by a description of several important factors that regulate bicarbonate transport in distal tubule, including the role of carbonic anhydrase and potassium depletion, and finally the participation of peptide hormones, in particular angiotensin II and arginine vasopressin. The analysis of the role of these factors is centered on microperfusion studies and on the analysis of cellular function based on cell pH and calcium modulation in renal cells in culture.

Peritubular AVP regulates bicarbonate reabsorption in cortical distal tubule via V(1) and V(2) receptors.

Peritubular arginine vasopressin (AVP) regulates bicarbonate reabsorption in the cortical distal tubule via V(1) and V(2) receptors. The dose-dependent effects of peritubular AVP on net bicarbonate reabsorption (J(HCO)) were evaluated by stationary microperfusion of in vivo early (ED; distal convoluted tubule) and late distal (LD; connecting tubule and initial collecting duct) segments of rat kidney, using double-barreled H(+)-sensitive, ion-exchange resin/reference (1 M KCl) microelectrodes. AVP (10(-11) M) perfused into peritubular capillaries increased J(HCO), compared with basal levels during intact capillary perfusion with blood, in ED and LD segments. AVP (10(-9) M) also increased J(HCO) in both segments, but the effect of AVP (10(-11) M) was significantly higher. A specificV(1)-receptor antagonist alone or with AVP (10(-11) or 10(-9) M) reduced J(HCO) below basal levels. A specific V(2)-receptor antagonist alone or plus AVP (10(-11) M) did not affect J(HCO) but increased AVP (10(-9) M)-mediated stimulation. 8-Bromoadenosine 3',5'-cyclic monophosphate alone reduced J(HCO) below basal levels and also reduced AVP (10(-11) M)-mediated stimulation. (Deamino-Cys(1), D-Arg(8)) vasopressin (a V(2)-selective agonist) also reduced J(HCO) below basal levels. These results show that peritubular AVP stimulates J(HCO) in ED and LD segments via basolateral V(1) receptors and that basolateral V(2) receptors have a dose-dependent inhibitory effect mediated by cAMP. The data also indicate that endogenous AVP stimulates distal J(HCO) via basolateral V(1) receptors.

Filtered load of buffer and renal H-ion secretion: mechanism of proximal tubule load dependence

Cuando la carga filtrada de "buffers" como bicarbonato o fosfato es incrementada por elevación del FG o de la concentración plasmática de "buffer", la reabsorción global de bicarbonato o la formación de acidez titulable son estimuladas. Lo mismo ocurre con la reabsorción proximal de bicarbonato durante la perfusión con concentraciones crecientes de este ion o cuando se eleva el flujo de fluido. En los últimos años, hemos estudiado los mecanismos de esta dependencia funcional. Observamos que el ritmo de reabsorción de bicarbonato es siempre proporcional a la concentración luminal de "buffer" cuando se inyecta una columna estacionaria de fluido en la luz tubular. La secreción de H**+ es tambiénn proporcional a nivels luminales de otros "buffers" distintos al bicarbonato. Empleando la técnica de pH-stat adaptada a túbulos renales, demostramos que la secreción proxim de H**+ depende del pH luminal y es independiente del sistema de "buffer" utilizado. Un análisis cinético de estos datos demuestra una relación no linear entre pH luminal y secreción de H**+, compatible con transporte mediado por "carrier"

Filtered load of buffer and renal H-ion secretion: mechanism of proximal tubule load dependence

Cuando la carga filtrada de "buffers" como bicarbonato o fosfato es incrementada por elevación del FG o de la concentración plasmática de "buffer", la reabsorción global de bicarbonato o la formación de acidez titulable son estimuladas. Lo mismo ocurre con la reabsorción proximal de bicarbonato durante la perfusión con concentraciones crecientes de este ion o cuando se eleva el flujo de fluido. En los últimos años, hemos estudiado los mecanismos de esta dependencia funcional. Observamos que el ritmo de reabsorción de bicarbonato es siempre proporcional a la concentración luminal de "buffer" cuando se inyecta una columna estacionaria de fluido en la luz tubular. La secreción de H**+ es tambiénn proporcional a nivels luminales de otros "buffers" distintos al bicarbonato. Empleando la técnica de pH-stat adaptada a túbulos renales, demostramos que la secreción proxim de H**+ depende del pH luminal y es independiente del sistema de "buffer" utilizado. Un análisis cinético de estos datos demuestra una relación no linear entre pH luminal y secreción de H**+, compatible con transporte mediado por "carrier" (AU)