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G Giebisch

Publications and source records attributed to G Giebisch.

At least 19 recordsLinked to original sources
PubMed1999

The relationship between distal tubular proton secretion and dietary potassium depletion: evidence for up-regulation of H+ -ATPase.

BACKGROUND: Dietary potassium depletion is associated with elevated plasma bicarbonate concentration and enhanced bicarbonate reabsorption in the distal tubule. The relationship between distal proton secretion and potassium status was investigated by in vivo microperfusion of the superficial distal tubule. METHODS: Experiments were performed on anaesthetized rats that had been maintained on either a low-potassium or control diet for 3-5 weeks prior to experimentation. The distal tubules were perfused at 10 nl/min with either a standard or a barium chloride-containing solution, and the late distal tubular transepithelial potential difference (Vte) and pH of the luminal fluid were recorded using a double-barrelled voltage and ion-sensitive microelectrode. RESULTS: In control rats, the Vte was -40.7+/-2.4 mV and the tubular fluid pH was 6.44+/-0.07; in potassium-depleted animals, the Vte was -15.0+/-1.4 mV and the pH was 6.76+/-0.03. The pH values in both groups of animals were significantly lower than would be predicted from the Vte and systemic pH for passive H+ distribution, indicating active proton secretion. Moreover, in hypokalaemic rats, this difference from predicted pH was significantly greater than in control animals (control = 0.27+/-0.06 vs. low-potassium = 0.46+/-0.03; P<0.01), suggesting enhanced active proton secretion. During perfusion with a solution containing BaCl2, the late distal tubule Vte became lumen positive in potassium-depleted rats, contrasting with an increased lumen negativity in potassium-replete controls. The barium-induced lumen-positive potential difference observed in the hypokalaemic rats was abolished by intravenous administration of acetazolamide. CONCLUSION: These data are consistent with enhanced electrogenic proton secretion (H+ -ATPase) during dietary potassium deprivation.

Animals↗
PubMed1999

Basolateral Na+ pump modulates apical Na+ and K+ conductances in rabbit cortical collecting ducts.

Previous studies indicated that an acute elevation of peritubular K+ enhances K+ secretion and Na+ reabsorption in the isolated perfused cortical collecting duct (CCD) from rabbit kidneys [S. Muto, G. Giebisch, and S. Sansom. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F108-F114, 1988]. To determine the underlying cellular mechanisms, we used microelectrode techniques to assess the membrane properties of collecting duct cells in isolated perfused CCDs of control and desoxycorticosterone acetate (DOCA)-treated rabbits following acute stimulation of the basolateral Na+-K+ pump by rapidly increasing the bath solution from 2.5 to 8.5 mM K+. This induced in both groups of tubules, first, a short-lasting hyperpolarization and, second, a sustained phase of depolarization of transepithelial, basolateral, and apical membrane voltages. Whereas the transepithelial conductance (GT) and fractional apical membrane resistance (fRA) remained unchanged during the initial phase of hyperpolarization, during the depolarization, GT increased and fRA decreased. Perfusion of the lumen with solutions containing either amiloride or Ba2+ attenuated the high K+-induced apical electrical changes, and basolateral strophanthidin abolished both apical and basolateral electrical responses during elevation of K+ in the bath. From these results we conclude the following: 1) acute elevation of basolateral K+ activates the basolateral Na+-K+ pump, which secondarily elevates the apical Na+ and K+ conductances; 2) DOCA pretreatment increases the basolateral K+ conductance and augments the response to the rise of K+ of both basolateral Na+-K+ pump activity and apical cation conductances.

Animals↗
PubMed1999

Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice.

NHE3 is the predominant isoform responsible for apical membrane Na(+)/H(+) exchange in the proximal tubule. Deletion of NHE3 by gene targeting results in an NHE3(-/-) mouse with greatly reduced proximal tubule HCO(-)(3) absorption compared with NHE3(+/+) animals (P. J. Schultheis, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull. Nature Genet. 19: 282-285, 1998). The purpose of the present study was to evaluate the role of other acidification mechanisms in mediating the remaining component of proximal tubule HCO(-)(3) reabsorption in NHE3(-/-) mice. Proximal tubule transport was studied by in situ microperfusion. Net rates of HCO(-)(3) (J(HCO3)) and fluid absorption (J(v)) were reduced by 54 and 63%, respectively, in NHE3 null mice compared with controls. Addition of 100 microM ethylisopropylamiloride (EIPA) to the luminal perfusate caused significant inhibition of J(HCO3) and J(v) in NHE3(+/+) mice but failed to inhibit J(HCO3) or J(v) in NHE3(-/-) mice, indicating lack of activity of NHE2 or other EIPA-sensitive NHE isoforms in the null mice. Addition of 1 microM bafilomycin caused a similar absolute decrement in J(HCO3) in wild-type and NHE3 null mice, indicating equivalent rates of HCO(-)(3) absorption mediated by H(+)-ATPase. Addition of 10 microM Sch-28080 did not reduce J(HCO3) in either wild-type or NHE3 null mice, indicating lack of detectable H(+)-K(+)-ATPase activity in the proximal tubule. We conclude that, in the absence of NHE3, neither NHE2 nor any other EIPA-sensitive NHE isoform contributes to mediating HCO(-)(3) reabsorption in the proximal tubule. A significant component of HCO(-)(3) reabsorption in the proximal tubule is mediated by bafilomycin-sensitive H(+)-ATPase, but its activity is not significantly upregulated in NHE3 null mice.

Absorption↗
PubMed1999

Origins of renal physiology in the USA.

Research in renal physiology began later in the USA than in Europe. Initial studies in the early 1900s dealt with the pathophysiology of renal failure. Micropuncture study of single nephron function was initiated in the laboratory of A.N. Richards in the early 1920s and with the later addition of microperfusion provided important insights into the site and mechanisms of solute transport. In parallel, with the leadership of Homer Smith, the development of noninvasive clearance methods to measure glomerular filtration rate and renal blood flow initiated a fruitful period of quantification of renal hemodynamics and of transport by the tubules. The steady progress of renal physiology in the USA owes much to the generous support of basic research by the National Institutes of Health. It is also worthy of mention that progress in renal physiology in the US owes much to work carried out in clinical departments and to the type of comparative studies exemplified by work at the Mt. Desert Island Biological Laboratory.

History, 19th Century↗
PubMed1999

Nongastric H+,K+-ATPase: cell biologic and functional properties.

Several members of the H+,K+-ATPase family of ion pumps participate in renal K transport. This class of P-type ATPases includes the gastric H+,K+-ATPase as well as a number of nongastric H+,K+-ATPase isoforms. Physiological studies suggest that these enzymes operate predominantly at the apical surfaces of tubule epithelial cells. Although much has been learned about the pattern of H+,K+-ATPase isoform expression and its response to stress, the functional and cell biologic attributes of these pumps remain largely unelucidated. We have studied the properties of renal H+,K+-ATPases both in vitro and in situ. Our analysis of ion fluxes driven by a nongastric H+,K+-ATPase isoform suggests that it exchanges Na (rather than H) for K under normal circumstances. Thus, the individual H+,K+-ATPase isoforms may make diverse contributions to renal cation transport. We find that the activities of renal H+,K+-ATPases in situ are regulated by endocytosis, which is mediated by an endocytosis signal in the cytoplasmic tail of the gastric H+,K+-ATPase beta-subunit. Transgenic mice expressing a version of this protein in which the signal has been disabled show constitutively active renal K resorption. The identities of the H+,K+-ATPase isoforms that are normally subject to endocytic regulation and the nature of the participating epithelial cell machinery have yet to be established.

Animals↗
PubMed1999

Physiological roles of renal potassium channels.

Potassium channels play an important role in a variety of transport operations in renal tubules. Considerable progress has been made in the analysis of their location and regulation by applying patch clamp techniques to native and cloned renal potassium channels. Potassium channels serve several functions, including the generation of the cell-negative potential and regulation of cell volume, particularly in the defense against cell swelling; they are also important for recycling and secretion of potassium. This review focuses on the properties of several potassium channels in the kidney and the important role in the regulation of potassium secretion in the distal nephron.

Adaptation, Physiological↗
PubMed1998

Contribution of Na+-H+ exchange to sodium reabsorption in the loop of henle: a microperfusion study in rats.

1. The contribution of apical Na+-H+ exchange to sodium reabsorption in the thick ascending limb of the loop of Henle (TALH) in vivo was examined in anaesthetized rats by perfusing loops of Henle of superficial nephrons with solutions containing the Na+-H+ exchange inhibitor, ethyl isopropyl amiloride (EIPA). 2. Using a standard perfusate, no statistically significant effect of EIPA on net sodium reabsorption (JNa) was detected. However, when sodium reabsorption in the pars recta of the proximal tubule was minimized by using a low-sodium perfusate, EIPA reduced JNa from 828 +/- 41 to 726 +/- 37 pmol min-1 (P < 0.05), indicating that apical Na+-H+ exchange can make a small contribution to net sodium reabsorption in the TALH in vivo. This contribution appears to be dependent on the bicarbonate load, since an increase in the latter led to an enhancement of EIPA-sensitive sodium transport. 3. Addition of the Na+-K+-2Cl- cotransport inhibitor, bumetanide, to the low-sodium perfusate reduced baseline JNa to 86 +/- 27 pmol min-1. In this setting, EIPA reduced JNa further, to -24 +/- 18 pmol min-1 (P < 0.05), an effect similar to that seen in the absence of bumetanide. This finding argues against previous suggestions (based on in vitro evidence) that inhibition of the Na+-K+-2Cl- cotransporter leads to an increase in apical Na+-H+ exchange in the TALH.

Amiloride↗
PubMed1998

Angiotensin II stimulates vesicular H+-ATPase in rat proximal tubular cells.

Two mechanisms of H+ ion secretion in the proximal tubule that mediate bicarbonate reabsorption have been identified: the brush border Na/H exchanger and electrogenic H+ ion secretion. Angiotensin II (AII) has been shown to be a regulator of the luminal Na+/H+ exchanger and the basolateral Na+/HCO3- cotransporter. In the present study, we examined the effects of AII on H+-ATPase activity in isolated proximal tubule fragments. H+-ATPase activity was assessed by monitoring intracellular pH after Na+ removal from the bath. In addition, we investigated the effects on pH recovery of the proton pump inhibitor bafilomycin A1, removal of Cl-, and of colchicine. pH was continuously measured with the pH-sensitive fluorescent dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Recovery of cell pH was observed in the absence of external Na+ and was significantly accelerated by AII. The AII-stimulated pH recovery was completely abolished by bafilomycin A1, by removal of Cl-, by NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate; a potent Cl- channel blocker], and by colchicine. We conclude from these studies that AII stimulates proton extrusion via H+-ATPase by a Cl--dependent process involving brush border insertion of vesicles. This process may contribute to up-regulation of HCO3- reabsorption along the proximal tubule when tubules are exposed to AII.

Angiotensin II↗
PubMed1998

Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger.

NHE3 is one of five plasma membrane Na+/H+ exchangers and is encoded by the mouse gene Slc9a3. It is expressed on apical membranes of renal proximal tubule and intestinal epithelial cells and is thought to play a major role in NaCl and HCO3- absorption. As the distribution of NHE3 overlaps with that of the NHE2 isoform in kidney and intestine, the function and relative importance of NHE3 in vivo is unclear. To analyse its physiological functions, we generated mice lacking NHE3 function. Homozygous mutant (Slc9a3-/-) mice survive, but they have slight diarrhoea and blood analysis revealed that they are mildly acidotic. HCO3- and fluid absorption are sharply reduced in proximal convoluted tubules, blood pressure is reduced and there is a severe absorptive defect in the intestine. Thus, compensatory mechanisms must limit gross perturbations of electrolyte and acid-base balance. Plasma aldosterone is increased in NHE3-deficient mice, and expression of both renin and the AE1 (Slc4a1) Cl-/HCO3- exchanger mRNAs are induced in kidney. In the colon, epithelial Na+ channel activity is increased and colonic H+,K+-ATPase mRNA is massively induced. These data show that NHE3 is the major absorptive Na+/H+ exchanger in kidney and intestine, and that lack of the exchanger impairs acid-base balance and Na+-fluid volume homeostasis.

Animals↗
PubMed1998

Renal potassium transport: mechanisms and regulation.

The regulation of potassium metabolism involves mechanisms for the appropriate distribution between the intra- and extracellular fluid compartments and for the excretion by the kidney. Clearance and single nephron studies show that renal excretion is determined by regulated potassium secretion and potassium reabsorption, respectively, in principal and intercalated cells of the distal nephron. Measurement of the electrochemical driving forces acting on potassium transport across individual cell membranes and characterization of several ATPases and potassium channels provide insights into the transport and regulation of renal potassium excretion.

Biological Transport↗
PubMed1998

Effect of metabolic acidosis on NaCl transport in the proximal tubule.

In metabolic acidosis, the capacity of the proximal tubule for bicarbonate absorption is enhanced, whereas NaCl reabsorption is inhibited. Recent evidence indicates that transcellular NaCl absorption in the proximal tubule is mediated by apical membrane Cl/formate exchange and Cl/oxalate exchange, in parallel with recycling of these organic anions. We evaluated whether the effect of metabolic acidosis to inhibit NaCl reabsorption in the proximal tubule is due at least in part to inhibition of organic anion-dependent NaCl transport in this nephron segment. Absorption rates of bicarbonate (JHCO3), chloride (JCl), and fluid (Jv) were measured in rat proximal tubule segments microperfused in situ. We confirmed that metabolic acidosis stimulates JHCO3 in tubules microperfused with 25 mM HCO3, pH 7.4. For measurements of JCl, tubules were microperfused with a low-bicarbonate (5 mM), high-chloride solution, simulating conditions in the late proximal tubule. Under these conditions, baseline JCl and Jv measured in the absence of formate and oxalate were not significantly different between control and acidotic rats. However, whereas addition of 50 ¿M formate or 1 ¿M oxalate to luminal and capillary perfusates markedly stimulated JCl and Jv in control rats, formate and oxalate failed to stimulate JCl and Jv in acidotic rats. We conclude that metabolic acidosis markedly downregulates organic anion-stimulated NaCl absorption, thereby allowing differential regulation of proximal tubule NaHCO3 and NaCl transport.

Absorption↗
PubMed1998

Partially active channels produced by PKA site mutation of the cloned renal K+ channel, ROMK2 (kir1.2).

The activity of the cloned renal K+ channel (ROMK2) is dependent on a balance between phosphorylation and dephosphorylation. There are only three protein kinase A (PKA) sites on ROMK2, with the phosphorylated residues being serine-25 (S25), serine-200 (S200), and serine-294 (S294) (Z.-C. Xu, Y. Yang, and S. C. Hebert. J. Biol. Chem. 271: 9313-9319, 1996). We previously mutated these sites from serine to alanine to study the contribution of each site to overall channel function. Here we have studied each of these single PKA site mutants using the single-channel configuration of the patch-clamp technique. Both COOH-terminal mutations at sites S200A and S294A showed a decreased open channel probability (Po), whereas the NH2-terminal mutation at site S25A showed no change in Po compared with wild-type ROMK2. The decrease in Po for the S200A and S294A mutants was caused by the additional presence of a long closed state. In contrast, the occurrence of the S25A channel was approximately 66% less, suggesting fewer active channels at the membrane. The S200A and S294A channels had different kinetics compared with wild-type ROMK2 channels, showing an increased occurrence of sublevels. Similar kinetics were observed when wild-type ROMK2 was excised and exposed to dephosphorylating conditions, indicating that these effects are specifically a property of the partially phosphorylated channel and not due to an unrelated effect of the mutation.

Animals↗
PubMed1998

A tyrosine-based signal regulates H-K-ATPase-mediated potassium reabsorption in the kidney.

Isoforms of the H-K-ATPase participate in active K resorption in the renal collecting tubule. The cytoplasmic tail of the beta-subunit of the gastric H-K-ATPase includes a 4 amino acid motif which is highly homologous to tyrosine-based endocytosis signals. We have generated transgenic mice expressing an H-K-ATPase beta-subunit in which the tyrosine residue in this sequence has been mutated to alanine. Mice expressing the mutated protein manifest constitutive hypersecretion of gastric acid, demonstrating that the beta-subunit tyrosine-based motif is required for the regulated endocytosis of the H-K pump and hence the cessation of gastric acid output. To test the possibility that the tyrosine-based sequence in the tail of the H-K-ATPase beta-subunit plays a role in regulating the function of renal H-K-ATPases, we examined renal K clearance in normal and in transgenic mice. Blood pressure, urine volume, glomerular filtration rate (GFR), plasma Na, and Na excretion are similar in control and transgenic mice. However, plasma K concentrations are significantly higher in transgenic mice (4.76 +/- 0.13 meq/l in transgenic and 4. 12 +/- 0.04 meq/l in control; n = 9, P < 0.05) and K excretion is lower in the transgenic animals (fractional excretion of K was 26.2 +/- 3.62% in transgenic and 50.1 +/- 4.78% in control; n = 9, P < 0. 01). These data suggest that the tyrosine-based signal in the cytoplasmic tail of the H-K-ATPase beta-subunit functions in the kidney as it does in the stomach to internalize H-K pump and thus inactivate pump function. Its elimination may result in the constitutive presence of the pump at the cell surface and lead to excessive urinary K reabsorption.

Animals↗
PubMed1998

pH-dependent modulation of the cloned renal K+ channel, ROMK.

pH is an important modulator of the low-conductance ATP-sensitive K+ channel of the distal nephron. To examine the mechanism of interaction of protons with the channel-forming protein, we expressed the cloned renal K channel, ROMK (Kir1.x), in Xenopus oocytes and examined the response to varied concentrations of protons both in the presence and in the absence of ATP. Initial experiments were performed on inside-out patches in the absence of ATP in Mg2+-free solution, which prevents channel rundown. A steep sigmoidal relationship was shown between bath pH and ROMK1 or ROMK2 channel function with intracellular acidification reducing channel activity. We calculated values for pK = 7.18 and 7.04 and Hill coefficients = 3.1 and 3.3, for ROMK1 and ROMK2, respectively. Intracellular acidification (pH 7.2) also increased the Mg-ATP binding affinity of ROMK2, resulting in a leftward shift of the relationship between ATP concentration and the reduction in channel activity. The K1/2 for Mg-ATP decreased from 2.4 mM at pH 7.4 to approximately 0.5 mM at pH 7.2. Mutation of lysine-61 to methionine in ROMK2, which abolishes pH sensitivity, modulated but did not eliminate the effect of pH on ATP inhibition of channel activity. We previously demonstrated that the putative phosphate loop in the carboxy terminus of ROMK2 is involved in ATP binding and channel inhibition [C. M. McNicholas, Y. Yang, G. Giebisch, and S. C. Hebert. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F275-F285, 1996]. Conceivably, therefore, protonation of the histidine residue within this region could alter net charge (i.e., positive shift) and increase affinity for the negatively charged nucleotide.

Acids↗
PubMed1997

Intracellular acidification induces Cl/HCO3 exchange activity in the basolateral membrane of beta-intercalated cells of the rabbit cortical collecting duct.

High speed video imaging microscopy and the pH-sensitive fluorophore2',7',-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) were used to examine acid-base functions of beta-intercalated cells of the rabbit cortical collecting duct. The presence of intercalated cells was established and the properties of apical and basolateral acid-base transporters assessed by monitoring cell pH during acid loading and luminal and basolateral ion substitutions. We showed that treatment of beta-intercalated cells with ammonium chloride (20 mm) induced a profound decrease of their intracellular pH from 6.98 +/- 5.93 +/- 0.08. pH recovery occurred after different lag periods ranging between 2 to 15 min (0.22 +/- 0. 04 dpH/dt). We demonstrated that this pH recovery mechanism was independent of basolateral Na+ and apical HCO-3 and K+. It was also not affected by apical and basolateral addition of NEM, by basolateral DIDS and by apical application of the H-KATPase inhibitor SCH28080. The process of pH recovery was however, critically dependent on basolateral HCO-3. These results are best explained by acid-induced insertion and/or activation of chloride-bicarbonate exchangers that are functional properties with their apical analogues.

Animals↗
PubMed1997

Nitric oxide links the apical Na+ transport to the basolateral K+ conductance in the rat cortical collecting duct.

We have used the patch clamp technique to study the effects of inhibiting the apical Na+ transport on the basolateral small-conductance K+ channel (SK) in cell-attached patches in cortical collecting duct (CCD) of the rat kidney. Application of 50 microM amiloride decreased the activity of SK, defined as nPo (a product of channel open probability and channel number), to 61% of the control value. Application of 1 microM benzamil, a specific Na+ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control value. In addition, benzamil reduced intracellular Na+ concentration from 15 to 11 mM. The effect of amiloride was not the result of a decrease in intracellular pH, since addition 50 microM 5-(n-ethyl-n-isopropyl) amiloride (EIPA), an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of amiloride depends on extracellular Ca2+ because removal of Ca2+ from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca2+, we observed that amiloride and benzamil significantly decreased intracellular Ca2+ in the Ca2+-containing solution but had no effect in a Ca2+-free bath. Furthermore, raising intracellular Ca2+ from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca2+ are responsible for the effects on SK activity of inhibition of the Na+ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and the function of the nNOS is Ca2+ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP-dependent pathways. Addition of 10 microM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 microM 8-bromoguanosine 3':5'-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca2+-free bath solution. We conclude that Ca2+-dependent NO generation mediates the effect of inhibiting the apical Na+ transport on the basolateral SK in the rat CCD.

Amiloride↗