A.A. Bernardo

Angiotensin II Stimulation of Renal Epithelial Cell Na/HCO3 Cotransport Activity: A Central Role for Src Family Kinase/Classic MAPK Pathway Coupling

Angiotensin II (AII) plays an important role in renal proximal tubular acidification via the costimulation of basolateral Na/HCO3 cotransporter (NBC) and apical Na/H exchanger (NHE) activities. These effects are mediated by specific G protein-coupled AII receptors, but their corresponding downstream effectors are incompletely defined. Src family tyrosine kinases (SFKs) contribute to the regulation of both transport activities by a variety of stimuli and are coupled to classic mitogen-activated protein kinase (MAPK) pathway activation in this cell type. We therefore examined these signaling intermediates for involvement in AII-stimulated NBC activity in cultured proximal tubule cells. Subpressor concentrations of AII (0.1 nM) increased NBC activity within minutes, and this effect was abrogated by selective antagonism of AT1 angiotensin receptors, SFKs, or the classic MAPK pathway. AII directly activated Src, as well as the proximal (Raf) and distal (ERK) elements of the classic MAPK module, and the activation of Src was prevented by AT1 receptor antagonism. An associated increase in basolateral membrane NBC1 content is compatible with the involvement of this proximal tubule isoform in these changes. We conclude that AII stimulation of the AT1 receptor increases NBC activity via sequential activation of SFKs and the classic MAPK pathway. Similar requirements for SFK/MAPK coupling in both cholinergic and acidotic costimulation of NBC and NHE activities suggest a central role for these effectors in the coordinated regulation of epithelial transport by diverse stimuli.

Effect of Renal Function on the Pharmacokinetics of Valsartan

SummaryThe effect of renal function on the pharmacokinetics of valsartan was investigated in this trial. In order to cover the full spectrum of renal function, a total of 19 subjects with normal renal function and various degrees of renal dysfunction, as determined by creatinine clearance (CLCR), were assigned to four groups: normal renal function (CLCR > 90 ml/min), and mild (CLCR 61 to 90 ml/min), moderate (CLCR 30 to 60 ml/min) and severe (CLCR < 30 ml/min) renal dysfunction. Creatinine clearance was determined following a 24-hour urine collection just prior to drug administration. Each subject received a single oral dose of 80mg of valsartan (capsule) after an overnight fast. Blood samples were collected at frequent intervals up to 48 hours postdose and plasma valsartan concentrations were determined. Pharmacokinetic parameters [area under the plasma concentration-time curve (AUC), maximum plasma valsartan concentration (Cmax), time to reach Cmax (tmax), and the terminal elimination half-life (t½)] were calculated. Statistical analysis using a cubic polynomial regression function was performed to examine a relationship between renal function and the pharmacokinetic parameters of valsartan.Scatter plots of pharmacokinetic parameters did not indicate any clear relationship with creatinine clearance. The regression coefficients of linear, quadratic and cubic terms for the AUC and Cmax of valsartan versus renal function were not significantly different from zero. Thus, the pharmacokinetics of valsartan did not correlate with renal function. In addition, no clinically significant adverse experiences were observed in this trial; the 80mg dose of valsartan was well tolerated. Based on these observations, there is no rationale for dosage adjustment of valsartan in patients with impaired renal function.

Renal cortical basolateral Na+/HCO3- cotransporter. I: Partial purification and reconstitution

The renal basolateral Na+/HCO3−cotransporter is the main system responsible for HCO3−transport from proximal tubule cells into the blood. The present study was aimed at purifying and functionally reconstituting the Na+/HCO3−cotransporter protein from rabbit renal cortex. Highly purified rabbit renal cortical basolateral membrane vesicles (hereafter designated as original basolateral membrane), enriched 12-fold in Na-K-ATPase, were solubilized in 2% octylglucoside, and then reconstituted in l-α-phosphatidylcholine (proteoliposomes). Na+/HCO3−cotransporter activity was assessed as the difference in 22Na uptake in the presence of HCO3−and gluconate. The activity of the Na+/HCO3−cotransporter was enhanced 18-fold in the solubilized protein reconstituted into proteoliposomes compared to the original basolateral membranes. The reconstituted solubilized purified protein exhibited kinetic properties similar to the cotransporter from original basolateral membranes. In addition, it was like the original cotransporter, inhibited by disulfonic stilbene SITS, and was eleetrogenic. The catalytic subunit of protein kinase A significantly inhibited Na+/HCO3−cotransporter activity in proteoliposomes. The octylglucoside-solubilized protein was further purified by hydroxylapatite column chromatography, and this resulted in an additional enhancement of Na+/HCO3−cotransporter activity of 80-fold over the original basolateral membranes. The fractions containing the highest activity were further processed by glycerol gradient centrifugation, resulting in a 124- to 300-fold increase in Na+/HCO3−cotransporter activity compared to the original basolateral membranes. SDS-PAGE analysis showed an enhancement of a protein doublet of 56 kD MW in the glycerol gradient fraction. Our results demonstrate that we have partially purified and reconstituted the renal Na+/HCO3−cotransporter and suggest that the 56 kD doublet protein may represent the Na+/HCO3−cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).

The role of phosphatidylinositol 3-kinase (PI3K) in CO2 stimulation of the Na+/HCO3- cotransporter (NBC).

The basolateral Na+/HCO3- cotransporter (NBC) is the major pathway for bicarbonate reabsorption in the renal proximal tubule cells. The cotransporter activity is enhanced by 10% CO2. Phosphatidylinositol 3-kinase (PI3K) has been shown to regulate the function and trafficking of cellular proteins by promoting their translocation to the plasma membrane. Therefore, we sought to examine the role of PI3K in CO2-mediated stimulation of NBC activity in OK cells. Our studies showed that wortmannin, a well-characterized PI3K inhibitor, had no effect on baseline NBC activity but prevented the stimulatory effect of 10% CO2. This effect was concentration-dependent and time-dependent. Another inhibitor of PI3K, LY294002, also prevented the CO2-mediated increase in NBC activity. CO2 stimulation of the cotransporter was paralleled by an increase in PI3K enzyme activity and this effect was blocked by wortmannin. Biotinylation studies also showed that 10% CO2 increased the immunoreactive NBC in the basolateral membranes and this was prevented by wortmannin. We previously showed that 10% CO2 stimulation of NBC activity involves the Src family kinase pathway. In the current studies, CO2 stimulation significantly increased Src phosphorylation and this effect was abrogated by wortmannin. In summary, CO2 stimulation of NBC is mediated at least in part by increased immunoreactive NBC protein in the basolateral membrane, a process which requires the interaction of PI3K with Src family kinase.

Renal cortical basolateral Na+/HCO 3 − cotransporter III. Evidence for a regulatory protein in the inhibitory effect of protein kinase A

The activity of the Na-H antiporter is inhibited by cyclic AMP-dependent protein kinase A (cAMP.PKA). The inhibitory effect of PKA on the Na-H antiporter is mediated through a regulatory protein that can be dissociated from the antiporter by limited protein digestion. PKA also inhibits the activity of the Na+/ HCO3−cotransporter. We investigated whether the activity of Na+/HCO3−cotransporter and the effect of PKA on this transporter may also be regulated by limited protein digestion. In rabbit renal cortical basolateral membranes (BLM) and in solubilized BLM reconstituted in liposomes (proteoliposomes), trypsin (100 μg) increased 22Na uptake in the presence of HCO3 but not in the presence of gluconate, indicating that trypsin does not alter diffusive 22Na uptake but directly stimulates the Na+/HCO3−cotransporter activity. In proteoliposomes phosphorylated with ATP, the catalytic subunit (CSU) of cAMP-PKA decreased the activity of the Na+/HCO3−cotransporter (expressed as nanomoles/mg protein/3s) from 23 ± 10 to 14 ± 6 (P < 0.01). In the presence of trypsin, the inhibitory effect of CSU of cAMP-PKA on the activity of Na+/HCO3−cotransporter was blunted. To identify a fraction that was responsible for the inhibitory effect of the CSU on the Na+/HCO3−cotransporter activity, solubilized proteins were separated by size exclusion chromatography. The effect of CSU of cAMP-PKA on the Na+/HCO3−cotransporter activity was assayed in proteoliposomes digested with trypsin with the addition of a fraction containing the 42 kDa protein (fraction S+) or without the 42 kDa protein (fraction S−). With the addition of fraction S−, the CSU of cAMP-PKA failed to inhibit the Na+/HCO3−cotransporter activity (control 27 ± 6, CSU 27 ± 3) while the addition of fraction S+ restored the inhibitory effect of CSU (27 ± 6 to 3 ± 0.3 P < 0.01). The CSU of cAMP-PKA phosphorylated several proteins in solubilized protein including a 42 kDa protein. Fluorescein isothiocyanate (FITC) labels components of the Na+/HCO3−cotransporter including the 56 kDa and 42 kDa proteins. In trypsin-treated solubilized protein the 42 kDa protein was not identified with FITC labeling. The results demonstrate that the activity of the Na+/HCO3−cotransporter is regulated by protein(s) which mediates the inhibitory effect of PKA. Limited protein digestion can dissociate this protein from the cotransporter.

Renal cortical basolateral Na+/HCO3- cotransporter. II: Detection of conformational changes with fluorescein isothiocyanate labeling

Fluorescein isothiocyanate (FITC) fluorescently labels amino groups and has been useful in detecting conformational changes in transport proteins through quenching or enhancement of the fluorescence signal upon exposure of protein to substrates. Solubilized renal basolateral membrane proteins, enriched in Na+/HCO3−cotransporter activity, were reconstituted into liposomes and treated with FITC or its nonfluorescent analogue PITC (phenyl isothiocyanate). In the absence of Na+ and HCO3−, incubation of proteoliposomes with PITC or FITC significantly inhibited cotransporter activity. However, in the presence of Na+ and HCO3−during labeling both agents failed to inhibit cotransporter activity, indicating that these probes interact specifically with the cotransporter. In the presence of the substrates Na+ and HCO3−, PITC binds covalently to amino groups unprotected by substrates leaving the Na+/HCO3−cotransporter available for specific labeling with FITC. Addition of NaHCO3 to FITC-labeled proteoliposomes resulted in a concentration-dependent enhancement of the fluorescence signal which was inhibited by pretreatment with 4,4′-diisothiocyanostilbene 2′,2-disulfonic acid (DIDS) prior to FITC labeling. SDS PAGE analysis of FITC-treated proteoliposomes showed the presence of two distinct fluorescent bands (approximate MW of 90 and 56 kD). In the presence of substrates, the fluorescence intensity of these bands was enhanced as confirmed by direct measurement of gel slice fluorescence. Thus, FITC detects conformational changes of the Na+/HCO3−cotransporter and labels proteins which may represent the cotransporter or components of this cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).

Renal Cortical Basolateral Na+/HCO− 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

Abstract. We have previously partially purified the basolateral Na+/HCO−3 cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na+/HCO−3 cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na+/HCO−3 cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na+/HCO−3 cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na+/HCO−3 cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO3 or Na2CO3) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na+/HCO−3 cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na+/HCO−3 cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na+/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues.

THE RENAL CORTICAL NA+/HCO3- COTRANSPORTER VI : THE EFFECT OF CHEMICAL MODIFICATION IN COTRANSPORTER ACTIVITY

Abstract. The Na+/HCO3− cotransporter is the main system that mediates bicarbonate removal out of the proximal tubule cell into the blood. We have previously partially purified this protein and showed that chemical modification of the α-amino groups by fluorescein isothiocyanate (FITC) inhibited the activity of the Na+/HCO3− cotransporter. The inhibition was prevented by the presence of Na and bicarbonate suggesting that this compound binds at or near the substrate transport sites of the cotransporter. We examined the effect of agents that modify the sulfhydryl group (dithiothreitol), carboxyl groups (n-n′dicyclohexyl carbodiimide) and tyrosine residues (p-nitrobenzene sulfonyl fluoride, n-acetyl imidazole and tetranitromethane) on the activity of the cotransporter to gain insight into the chemical residues which may be important for transport function. The sulfhydryl residues modifier, carboxyl group modifier, and tyrosine modifier significantly inhibited bicarbonate dependent 22Na uptake in basolateral membranes by 50–70% without altering the 22Na uptake in the presence of gluconate indicating that these agents directly affected the cotransporter without affecting diffusive sodium uptake. The effect of the tyrosine modifier n-acetylimidazole was not prevented by the presence of Na and bicarbonate suggesting that the tyrosine residues are not at the substrate binding sites. To determine the presence and role of glycosylation on the Na+/HCO3− cotransporter protein, we examined the effects of different glycosidases (endoglycosidase F and H, N-glycosidase F, O-glycanase) on the cotransporter activity. All glycosidases caused a significant 50–80% inhibition of cotransporter activity. These data demonstrate that N-glycosylation as well as O-glycosylation are important for the function of the Na+/HCO3− cotransporter protein. Taken together, these results suggest that chemical modifiers of tyrosine, carboxyl and sulfhydryl groups as well as glycosylation are important for expression of full functional activity of the cotransporter.