IntroductionThe renal proximal tubule reabsorbs more than 80% of the filtered bicarbonate load. The principal transport systems (1-3) responsible for transmembrane movement of hydrogen and bicarbonate ions in the proximal tubule cells are the apical Na + /H -exchanger type 3 isoform (NHE3) and the basolateral Na + /HCO 3 -cotransporter (NBC). It might be predicted that systemic and proximal tubule intracellular acid-base homeostasis requires that NHE3 and NBC function in a coordinate fashion. The uniformly parallel regulation of both transporters under a variety of physiologic conditions is consistent with such a hypothesis. NBC activity is enhanced by metabolic acidosis and inhibited by metabolic alkalosis (4). Similarly, chronic hypercapnia increases NBC activity, whereas chronic respiratory alkalosis has the opposite effect (5). NBC activity is also modulated by angiotensin II and parathyroid hormone (6, 7). Under identical conditions, NHE3 activity varies in parallel, suggesting that the activities of the 2 systems may be coordinately regulated (4). The parallel regulation of these transporters has also recently been extended to the level of specific regulatory protein kinases (8, 9). Phosphorylation of brush-border membrane (BBM) and basolateral membrane (BLM) proteins by either protein kinase A (PKA) or calcium calmodulin multifunction protein kinase II (Ca-CAMK II) is inhibitory for both NHE and NBC activities in the respective membranes, whereas protein kinase C (PKC) is stimulatory (8). In the renal proximal tubule, the activities of the basolateral Na + /HCO 3 -cotransporter (NBC) and the apical Na + /H + exchanger (NHE3) uniformly vary in parallel, suggesting that they are coordinately regulated. PKA-mediated inhibition of NHE3 is mediated by a PDZ motif-containing protein, the Na + /H + exchanger regulatory factor (NHE-RF). Given the common inhibition of these transporters after protein kinase A (PKA) activation, we sought to determine whether NHE-RF also plays a role in PKA-regulated NBC activity. Renal cortex immunoblot analysis using anti-peptide antibodies directed against rabbit NHE-RF demonstrated the presence of this regulatory factor in both brush-border membranes (BBMs) and basolateral membranes (BLMs). Using a reconstitution assay, we found that limited trypsin digestion of detergent solubilized rabbit renal BLM preparations resulted in NBC activity that was unaffected by PKA activation. Co-reconstitution of these trypsinized preparations with a recombinant protein corresponding to wild-type rabbit NHE-RF restored the inhibitory effect of PKA on NBC activity in a concentration-dependent manner. NBC activity was inhibited 60% by 10 -8 M NHE-RF; this effect was not observed in the absence of PKA. Reconstitution with heat-denatured NHE-RF also failed to attenuate NBC activity. To establish further a physiologic role for NHE-RF in NBC regulation, the renal epithelial cell line B-SC-1, which lacks detectable endogenous NHE-RF expression, was engineered to express stably an NHE-RF transgene. NHE-...

Studies were performed to determine the effect of protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and calcium-phospholipid-dependent protein kinase on Na+/H+ exchange activity. Proteins from the apical membrane of the proximal tubule of the rabbit kidney were solubilized in octyl glucoside and incubated in phosphorylating solutions containing the protein kinase. 22Na+ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. Calcium-calmodulin-dependent multifunction protein kinase II inhibited the amiloride-sensitive component of proton gradient-stimulated Na+ uptake in a dose-dependent manner. The inhibitory effect of this kinase had an absolute requirement for calmodulin, Ca2+, and ATP. Calcium-phospholipid-dependent protein kinase stimulated the amiloride-sensitive component of proton gradient-stimulated Na+ uptake in a dose-dependent manner. The stimulating effect of this kinase had an absolute requirement for ATP, Ca2+, and an active phorbol ester. These experiments indicate that Na+/H+ exchange activity of proteoliposomes reconstituted with proteins from renal brush-border membranes are inhibited by protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and stimulated by that mediated by calcium-calmodulin-dependent protein kinase.

To study the relation between the human Na'-H' exchanger (NHE-1) and the renal brush border membrane (BBM) Na'-H+ exchanger, polyclonal antibodies to synthetic peptides representing a putative external (Ab-E) and an internal cytosolic domain (Ab-I) of human NHE-1 were generated in rabbits. Western immunoblot analyses indicated that both antibodies recognized a 97-kD protein in rabbit renal BBM but not basolateral membranes (BLM). Octyl glucoside-extracted rabbit renal BBM proteins also contained the 97-kD polypeptide as did a fraction eluted from an anion-exchange column with 0.2 M NaCl (fraction A). A fraction eluting between 0.2 and 0.4 M NaCI (fraction B) did not contain this protein. Prior reconstitution studies have indicated that Na'-H' exchange activity is higher significantly in fraction B than fraction A. Administration of NH4CJ for 3-7 d to rabbits, a stimulus known to increase renal BBM Na'-H+ exchange activity, did not result in a change in expression of the 97-kD protein in either renal BBM or BLM.The results indicate that affinity-purified polyclonal antibodies to two separate domains of the human Na'-H+ exchanger recognize a 97-kD protein in rabbit renal BBM but not BLM. The dissociation between recognition of the 97-kD protein using antibodies and the majority of functional Na'-H' exchange activity after chromatographic fractionation ofsolubilized BBM proteins and in native BBM after administration of NH4Cl suggests that rabbit renal BBM contains more than one form of Na '-H' exchanger. (J. Clin. Invest. 1993. 91:2097-2102