NHERF-1 uniquely transduces the cAMP signals that inhibit sodium-hydrogen exchange in mouse renal apical membranes

Sodium-hydrogen exchanger regulatory factor isoform-1 (NHERF-1) and NHERF-2 are two structurally related PDZ-domain-containing protein adapters that effectively transduce cyclic AMP (cAMP) signals that inhibit NHE3, the sodium-hydrogen exchanger isoform present at the apical surface of kidney and gut epithelia. The mouse renal proximal tubule expresses both NHERF isoforms, suggesting their redundant functions as regulators of renal electrolyte metabolism. To define the role of NHERF-1 in the physiological control of NHE3, we analyzed NHE3 activity in isolated brush border membrane (BBM) preparations from renal proximal tubules of wild-type (WT) and NHERF-1 (-/-) mice. Basal Na(+)-H(+) exchange was indistinguishable in BBMs from WT and NHERF-1 (-/-) mice (0.96+/-0.08 and 0.95+/-0.10 nmol/mg protein/10 s, respectively). Activation of membrane bound cAMP-dependent protein kinase (PKA) by cAMP inhibited NHE3 activity in WT BBMs (0.55+/-0.07 nmol/mg protein/10 s or 40+/-9%, P<0.01) but had no discernible effect on Na(+)-H(+) exchange in the NHERF-1 (-/-) BBM (0.97+/-0.07 nmol/mg protein/10 s; P=not significant). This was associated with a significant decrease in cAMP-stimulated phosphorylation of NHE3 immunoprecipitated from solubilized NHERF-1 (-/-) BBMs. As the protein levels for NHE3, NHERF-2, PKA and ezrin were not changed in the NHERF-1 (-/-) BBMs, the data suggest a unique role for NHERF-1 in cAMP-mediated inhibition of NHE3 activity in the renal proximal tubule of the mouse.     

Specific association of megalin and the Na+/H+ exchanger isoform NHE3 in the proximal tubule.

We investigated whether the renal brush border Na+/H+ exchanger NHE3 exists in assemblies with other proteins in native kidney membranes. To this end we generated monoclonal antibodies (mAbs) against affinity purified NHE3 protein complexes. Hybridomas were selected based on ability to immunoprecipitate NHE3. One of the resulting mAbs (10A3) labeled a high molecular mass (>200 kDa) protein and stained primarily the coated pit region of the proximal tubule in a manner similar to that described for megalin (gp330). We then confirmed that both mAb 10A3 and a known anti-megalin mAb immunoprecipitated and immunoblotted the same protein, namely megalin. mAb 10A3 specifically co-precipitated NHE3 but not villin or NaPi-2 from solubilized renal membranes, indicating specificity of the NHE3-megalin interaction. When immunoprecipitations were performed using either 10A3 or anti-NHE3 mAb 2B9 after separation of solubilized renal proteins by sucrose velocity gradient centrifugation, we found that NHE3 exists in two states with distinct sedimentation coefficients, a 9.6 S megalin-free form and a 21 S megalin-bound form, and that when NHE3 assembles with megalin, epitopes within the carboxyl-terminal 131 amino acids of NHE3 are blocked. Taken together, these findings indicate that a significant pool of NHE3 exists as a multimeric complex with megalin in the brush border of the proximal tubule.     

Active (9.6 s) and inactive (21 s) oligomers of NHE3 in microdomains of the renal brush border

We have previously shown that Na(+)-H(+) exchanger isoform NHE3 exists as both 9.6 and 21 S (megalin-associated) oligomers in the renal brush border. To characterize the oligomeric forms of the renal brush border Na(+)-H(+) exchanger in more detail, we performed membrane fractionation studies. We found that similar amounts of NHE3 were present in microvilli and a nonmicrovillar membrane domain of high density (dense vesicles). Horseradish peroxidase-labeled endosomes were not prevalent in the dense membrane fraction. However, megalin, which localizes primarily to the intermicrovillar microdomain of the brush border, was enriched in the dense vesicles, implicating this microdomain as the likely source of these membranes. Immunolocalization of NHE3 confirmed that a major fraction of the transporter colocalized with megalin in the intermicrovillar region of the brush border. Immunoprecipitation studies demonstrated that in microvilli the majority of NHE3 was not bound to megalin, while in the dense vesicles most of the NHE3 coprecipitated with megalin. Moreover, sucrose velocity gradient centrifugation experiments revealed that most NHE3 in microvilli sedimented with an S value of 9.6, while the S value of NHE3 in dense vesicles was 21. Finally, we examined the functional state of NHE3 in both membrane fractions. As assayed by changes in acridine orange fluorescence, imposing an outwardly directed Na(+) gradient caused generation of an inside acid pH gradient in the microvilli, indicating Na(+)-H(+) exchange activity, but not in the dense vesicles. Taken together, these data demonstrate that renal brush border NHE3 exists in two oligomeric states: a 9.6 S active form present in microvilli and a 21 S, megalin-associated, inactive form in the intermicrovillar microdomain of the apical plasma membrane. Thus, regulation of renal brush border Na(+)-H(+) exchange activity may be mediated by shifting the distribution between these forms of NHE3.     

Detection of apical Na(+)/H(+) exchanger activity inhibition in proximal tubules induced by acute hypertension

We previously showed that acute arterial hypertension induces an inhibition of fluid and NaCl reabsorption in proximal tubules of Sprague-Dawley rats, which is associated with a rapid reversible internalization of apical Na(+)/H(+) exchanger in brush border. To determine whether there is a corresponding inhibition of apical Na(+)/H(+) exchanger activity in proximal tubules to account for the reduced tubular reabsorption, an instrument capable of measuring intracellular pH (pH(i)) ratiometrically and repeatedly on the surface of kidney with high temporal resolution is required. We report the design and validation of such a fluorimetric system based on two ultraviolet nitrogen-pulsed lasers and a photomultiplier. pH(i) of proximal tubules in situ was measured with pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein at 5 Hz. Using the initial rate of change of pH(i) (dpH(i)/dt) during luminal Na(+) removal as an index of apical Na(+)/H(+) exchanger activity, the exchanger activity was found to be reduced by 52 +/- 11% (n = 14, P < 0.05) compared with the baseline after 20 min of induced acute hypertension. The inhibition of Na(+)/H(+) exchange activity was alleviated when the blood pressure was returned to prehypertensive level. These observations indicate that acute changes in arterial pressure can reversibly inhibit apical Na(+)/H(+) exchanger activity, which might contribute to pressure natriuresis in proximal tubule.     

Ion exchangers mediating NaCl transport in the renal proximal tubule

We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. Studies of isolated brush-border membrane vesicles confirmed the presence of Na+-H+ exchange and identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanisms of uphill Cl- entry. We found that formate and oxalate each stimulate NaCl absorption in microperfused proximal tubules. Stimulation of NaCl absorption by formate was blocked by the Na+-H+-exchange inhibitor EIPA, whereas stimulation by oxalate was blocked by omission of sulfate from the perfusion solutions. These observations were consistent with recycling of formate from lumen to cell by H+-coupled formate transport in parallel with Na+-H+ exchange and recycling of oxalate by oxalate-sulfate exchange in parallel with Na+-sulfate cotransport. Using isoform-specific antibodies, we found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the loop of Henle. The inhibitor sensitivity of Na+-H+ exchange in renal brush-border vesicles and of HCO3- absorption in microperfused tubules suggested that NHE3 is responsible for most, if not all, apical membrane Na+-H+ exchange in the proximal tubule. The role of NHE3 in mediating proximal tubule HCO3- absorption and formate-dependent Cl- absorption was confirmed by studies in NHE3 null mice. Finally, we cloned and functionally expressed CFEX, an anion transporter expressed on the apical surface of proximal tubule cells and capable of mediating Cl(-)-formate exchange.     

Acid increases NHE8 surface expression and activity in NRK cells

We previously demonstrated that there is a paucity of brush-border membrane NHE3 in neonates, the predominant Na(+)/H(+) exchanger in the adult proximal tubule, while NHE8 is relatively highly expressed in neonates compared with adults. We recently showed that metabolic acidosis in neonatal rodents can increase brush-border membrane NHE8 protein expression and Na(+)/H(+) exchange activity. To further examine the regulation of NHE8 by acid, we incubated NRK cells, which express NHE8 but not NHE3, with either acid or control media (6.6 vs. 7.4). There was an increase in Na(+)/H(+) exchanger activity within 6 h of incubation with acid media assessed as the rate of sodium-dependent recovery of pH from an acid load (dpH(i)/dt). The acid stimulation persisted for at least 24 h. The increase in Na(+)/H(+) exchange activity was paralleled by an increase in surface expression of NHE8, assessed by surface biotinylation and streptavidin precipitation. The increase in both apical membrane NHE8 protein expression and Na(+)/H(+) exchange activity with pH 6.6 media compared with 7.4 media was not affected by actinomycin D or cycloheximide consistent with an increase in surface expression independent of mRNA or protein synthesis. Furthermore, there was no increase in total cellular NHE8 protein abundance or mRNA abundance with acid media. Finally, we demonstrate that the increase in surface expression of NHE8 with acid media was blocked by colchicine and cytochalasin D and mediated by acid increasing the rate of exocytosis. In conclusion, NHE8 surface expression and activity are regulated by acid media by increasing the rate of trafficking to the apical membrane.     

Lysophosphatidic acid induces exocytic trafficking of Na(+)/H(+) exchanger 3 by E3KARP-dependent activation of phospholipase C

Lysophosphatidic acid (LPA) stimulates Na(+)/H(+) exchanger 3 (NHE3) activity in opossum kidney proximal tubule (OK) cells by increasing the apical membrane amount of NHE3. This occurs by stimulation of exocytic trafficking of NHE3 to the apical plasma membrane by an E3KARP-dependent mechanism. However, it is still unclear how E3KARP leads to the LPA-induced exocytosis of NHE3. In the current study, we demonstrate that stable expression of exogenous E3KARP increases LPA-induced phospholipase C (PLC) activation and subsequent elevation of intracellular Ca(2+) in opossum kidney proximal tubule (OK) cells. Pretreatment with U73122, a PLC inhibitor, prevented the LPA-induced NHE3 activation and the exocytic trafficking of NHE3. To understand how the elevation of intracellular Ca(2+) leads to the stimulation of NHE3, we pretreated OK cells with BAPTA-AM, an intracellular Ca(2+) chelator. BAPTA-AM completely blocked the LPA-induced increase of NHE3 activity and surface NHE3 amount by decreasing the LPA-induced exocytic trafficking of NHE3. Pretreatment with GF109203X, a PKC inhibitor, did not affect the percent of LPA-induced NHE3 activation and increase of surface NHE3 amount. From these results, we suggest that E3KARP plays a necessary role in LPA-induced PLC activation, and that PLC-dependent elevation of intracellular Ca(2+) but not PKC activation is necessary for the LPA-induced increase of NHE3 exocytosis.     

Differential renal distribution of NHERF isoforms and their colocalization with NHE3, ezrin, and ROMK

Na(+)/H(+) exchanger regulatory factor (NHERF) and NHERF2 are PDZ motif proteins that mediate the inhibitory effect of cAMP on Na(+)/H(+) exchanger 3 (NHE3) by facilitating the formation of a multiprotein signaling complex. With the use of antibodies specific for NHERF and NHERF2, immunocytochemical analysis of rat kidney was undertaken to determine the nephron distribution of both proteins and their colocalization with other transporters and with ezrin. NHERF was most abundant in apical membrane of proximal tubule cells, where it colocalized with ezrin and NHE3. NHERF2 was detected in the glomerulus and in other renal vascular structures. In addition, NHERF2 was strongly expressed in collecting duct principal cells, where it colocalized with ROMK. These results indicate a striking difference in the nephron distribution of NHERF and NHERF2 and suggests NHERF is most likely to be the relevant biological regulator of NHE3 in the proximal tubule, while NHERF2 may interact with ROMK or other targets in the collecting duct. The finding that NHERF isoforms occur in different cell types suggests that NHERF and NHERF2 may subserve different functions in the kidney.     

Upregulation of apical NHE3 in renal OK cells overexpressing the rodent alpha(1)-subunit of the Na(+) pump

Vectorial Na(+) reabsorption across the proximal tubule is mediated by apical entry of Na(+), primarily via Na(+)/H(+) exchanger isoform 3 (NHE3), and basolateral extrusion via the Na(+) pump (Na(+)-K(+)-ATPase). We hypothesized that regulation of Na(+) reabsorption should involve not only the activity of the basolateral Na(+)-K(+)-ATPase, but also the apical NHE3, in a concerted manner. To generate a cell line that overexpresses Na(+)-K(+)-ATPase, opossum kidney (OK) cells were transfected with the rodent Na(+)-K(+)-ATPase alpha(1)-subunit (pCMV ouabain vector), and native cells were used as a control. The existence of distinct functional classes of Na(+)-K(+)-ATPase in wild-type and transfected cells was confirmed by the inhibition profile of Na(+)-K(+)-ATPase activity by ouabain. In contrast to wild-type cells, transfected cells exhibited two IC(50) values for ouabain: the first value was similar to the IC(50) of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum alpha(1)-isozymes. It is shown that transfection of OK cells with Na(+)-K(+)-ATPase increased Na(+)-K(+)-ATPase and NHE3 activities. This was associated with overexpression of the Na(+)-K(+)-ATPase alpha(1)-subunit and NHE3 in transfected OK cells. The abundance of the Na(+)-K(+)-ATPase beta(1)-subunit was slightly lower in transfected OK cells. In conclusion, the increase in expression and function of Na(+)-K(+)-ATPase in cells transfected with the rodent Na(+) pump alpha(1)-subunit cDNA is expected to stimulate apical Na(+) influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.     

Angiotensin II and proximal tubule sodium transport.

As a target site for angiotensin II (A-II), renal proximal tubule is unique in that it may be equipped with a local A-II generating system and that both basolateral and apical membranes may be accessible for A-II's action. We have recently conducted studies to examine these possibilities. With in vitro cultured proximal tubular cells, we have demonstrated de novo synthesis of angiotensinogen and renin. With isolated renal brush border membrane (BBM), we have confirmed the presence of A-II receptors and found that A-II directly stimulated BBM Na(+)-H+ exchange. In search of the signal transduction mechanism, we have found that A-II also activated BBM phospholipase A2 (PLA) and that BBM contained a pertussis toxin-sensitive guanine nucleotide binding protein (G-protein) which mediates the effects of A-II. Further studies showed that prevention of PLA activation abolished A-II's effect on Na(+)-H+ exchange, and that activation of PLA by mellitin and addition of arachidonic acid similarly enhanced Na(+)-H+ exchange activity, suggesting that PLA activation may mediate the stimulatory effect of A-II on Na(+)-H+ exchange. These results thus indicate that a local signal transduction mechanism involving G-protein mediated PLA activation exists in renal BBM which mediates A-II's effect on Na(+)-H+ exchange. Taken together, we propose that, independent of A-II in the circulation, local luminal A-II may serve as an important regulatory system on sodium transport in renal proximal tubule.     

Indirect regulation of the intestinal H+-coupled amino acid transporter hPAT1 (SLC36A1)

A H(+)-coupled amino acid transporter has been characterised functionally at the brush border membrane of the human intestinal cell line Caco-2. This carrier, hPAT1 (human Proton-coupled Amino acid Transporter 1) or SLC36A1, has been identified recently at the molecular level and hPAT1 protein is localised to the brush border membrane of human small intestine. hPAT1 transports both amino acids (e.g., beta-alanine) and therapeutic agents (e.g., D-cycloserine). In human Caco-2 cells, hPAT1 function (H(+)/amino acid symport) is associated with a decrease in intracellular pH (pH(i)), which selectively activates the Na(+)/H(+) exchanger NHE3, and thus maintains pH(i) and the driving force for hPAT1 function (the H(+) electrochemical gradient). This study provides the first evidence for regulation of hPAT1 function. Activation of the cAMP/protein kinase A pathway in Caco-2 cell monolayers either using pharmacological tools (forskolin, 8-br-cAMP, [(11,22,28)Ala]VIP) or physiological activators (the neuropeptides VIP and PACAP) inhibited hPAT1 function (beta-alanine uptake) at the apical membrane. Under conditions where NHE3 is inactive (the absence of Na(+), apical pH 5.5, the presence of the NHE3 inhibitor S1611) no regulation of beta-alanine uptake is observed. Forskolin and VIP inhibit pH(i) recovery (NHE3 function) from beta-alanine-induced intracellular acidification. Immunocytochemistry localises NHERF1 (NHE3 regulatory factor 1) to the apical portion of Caco-2 cells where it will interact with NHE3 and allow PKA-mediated phosphorylation of NHE3. In conclusion, we have shown that amino acid uptake via hPAT1 is inhibited by activators of the cAMP pathway indirectly through inhibition of NHE3 activity.     

Aldosterone stimulates activity and surface expression of NHE3 in human primary proximal tubule epithelial cells (RPTEC).

The steroid hormone aldosterone is a major regulator of extracellular volume and blood pressure. Aldosterone effectors are for example the epithelial Na(+) channel (ENaC), the Na(+)-K(+)-ATPase and the proximal tubule Na(+)/H(+) exchanger isoform 3 (NHE3). The aim of this study was to investigate whether aldosterone acts directly on proximal tubule cells to stimulate NHE3 and if so whether the EGF-receptor (EGFR) is involved. For this purpose, primary human renal proximal tubule cells were exposed to aldosterone. NHE3 activity was determined from Na(+)- dependent pH-recovery, NHE3 surface expression was determined by biotinylation and immunoblotting. EGFR-expression was assessed by ELISA. pH(i)- measurements revealed an aldosterone-induced increase in NHE3 activity, which was inhibited by the mineralocorticoid receptor blocker spironolactone and by the EGFR-kinase inhibitor AG1478. Immunoprecipitation and immunoblot analysis showed an aldosterone-induced increase in NHE3 surface expression, which was also inhibited by spironolactone and AG1478. Furthermore, aldosterone enhanced EGFR-expression. In conclusion, aldosterone stimulates NHE3 in human proximal tubule cells. The underlying mechanisms include AG1478 inhibitable kinase and are paralleled by enhanced EGFR expression, which could be compatible with EGF-receptor-pathway-dependent surface expression and activity of NHE3 in human primary renal proximal tubule epithelial cells.     

Acute regulation of Na+/H+ exchanger NHE3 by parathyroid hormone via NHE3 phosphorylation and dynamin-dependent endocytosis

Parathyroid hormone (PTH) is a potent inhibitor of mammalian renal proximal tubule Na(+) transport via its action on the apical membrane Na(+)/H(+) exchanger NHE3. In the opossum kidney cell line, inhibition of NHE3 activity was detected from 5 to 45 min after PTH addition. Increase in NHE3 phosphorylation on multiple serines was evident after 5 min of PTH, but decrease in surface NHE3 antigen was not detectable until after 30 min of PTH. The decrease in surface NHE3 antigen was due to increased NHE3 endocytosis. When endocytic trafficking was arrested with a dominant negative dynamin mutant (K44A), the early inhibition (5 min) of NHE3 activity by PTH was not affected, whereas the late inhibition (30 min) and decreased surface NHE3 antigen induced by PTH were abrogated. We conclude that PTH acutely inhibits NHE3 activity in a biphasic fashion by NHE3 phosphorylation followed by dynamin-dependent endocytosis.     

Dopamine acutely stimulates Na+/H+ exchanger (NHE3) endocytosis via clathrin-coated vesicles: dependence on protein kinase A-mediated NHE3 phosphorylation

Dopamine (DA) is a key hormone in mammalian sodium homeostasis. DA induces natriuresis via acute inhibition of the renal proximal tubule apical membrane Na(+)/H(+) exchanger NHE3. We examined the mechanism by which DA inhibits NHE3 in a renal cell line. DA acutely decreases surface NHE3 antigen in dose- and time-dependent fashion without altering total cellular NHE3. Although DA(1) receptor agonist alone decreases surface NHE3, simultaneous DA(2) agonist synergistically enhances the effect of DA(1). Decreased surface NHE3 antigen, caused by stimulation of NHE3 endocytosis, is dependent on intact functioning of the GTPase dynamin and involves increased binding of NHE3 to the adaptor protein AP2. DA-stimulated NHE3 endocytosis can be blocked by pharmacologic or genetic protein kinase A inhibition or by mutation of two protein kinase A target serines (Ser-560 and Ser-613) on NHE3. We conclude that one mechanism by which DA induces natriuresis is via protein kinase A-mediated phosphorylation of proximal tubule NHE3 leading to endocytosis of NHE3 via clathrin-coated vesicles.     

Tissue-specific regulation of sodium/proton exchanger isoform 3 activity in Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) null mice. cAMP inhibition is differentially dependent on NHERF1 and exchange protein directly activated by cAMP in ileum versus proximal tubule

The multi-PDZ domain containing protein Na(+)/H(+) Exchanger Regulatory Factor 1 (NHERF1) binds to Na(+)/H(+) exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1(-/-) mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1(-/-) ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1(-/-) mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2'O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1(-/-) mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC.     

High affinity binding of amiloride analogs at an internal site in renal microvillus membrane vesicles.

Amiloride analogs with hydrophobic substitutions on the 5-amino nitrogen atom are relatively high affinity inhibitors of the plasma membrane Na(+)-H+ exchanger. We demonstrated that a high affinity-binding site for [3H]5-(N-methyl-N-isobutyl)amiloride ([3H]MIA) (Kd = 6.3 nM, Bmax = 1.2 pmol/mg of protein) is present in microvillus membrane vesicles but not in basolateral membrane vesicles isolated from rabbit renal cortex, in accord with the known membrane localization of the Na(+)-H+ exchanger in this tissue. The rank order potency for inhibition of microvillus membrane [3H]MIA binding by amiloride analogs was: MIA (I50 approximately 10 nM) greater than amiloride (I50 approximately 200 nM) greater than benzamil (I50 approximately 1200 nM). This correlated with a qualitatively similar rank order potency for inhibition of Na(+)-H+ exchange: MIA (I50 approximately 4 microM) greater than amiloride (I50 approximately 15 microM) greater than benzamil (I50 approximately 100 microM), but did not correlate with the rank order potency for inhibition of the organic cation-H+ exchanger in microvillus membrane vesicles: MIA approximately benzamil (I50 approximately 0.5 microM) greater than amiloride (I50 approximately 10 microM). However, tetraphenylammonium, an inhibitor of organic cation-H+ exchange, inhibited the rate of [3H]MIA binding without an effect on equilibrium [3H]MIA binding; the dissociation of bound [3H]MIA was inhibited by preloading the membrane vesicles with tetraphenylammonium. These findings indicated that high affinity [3H]MIA binding to renal microvillus membrane vesicles takes place at an internal site to which access is rate-limited by the tetraphenylammonium-sensitive organic cation transporter. Equilibrium [3H]MIA binding was inhibited by H+ but was unaffected by concentrations of Na+ or Li+ that saturate the external transport site of the Na(+)-H+ exchanger. Binding of MIA to its high affinity binding site had no effect on the rate of Na(+)-H+ exchange. This study suggests that the renal Na(+)-H+ exchanger has a high affinity internal binding site for amiloride analogs that is distinct from the external amiloride inhibitory site.     

Cargo-selective apical exocytosis in epithelial cells is conducted by Myo5B,Slp4a,Vamp7, and Syntaxin 3

Mutations in the motor protein Myosin Vb (Myo5B) or the soluble NSF attachment protein receptor Syntaxin 3 (Stx3) disturb epithelial polarity and cause microvillus inclusion disease (MVID), a lethal hereditary enteropathy affecting neonates. To understand the molecular mechanism of Myo5B and Stx3 interplay, we used genome editing to introduce a defined Myo5B patient mutation in a human epithelial cell line. Our results demonstrate a selective role of Myo5B and Stx3 for apical cargo exocytosis in polarized epithelial cells. Apical exocytosis of NHE3, CFTR (cystic fibrosis transmembrane conductance regulator), and GLUT5 required an interaction cascade of Rab11, Myo5B, Slp4a, Munc18-2, and Vamp7 with Stx3, which cooperate in the final steps of this selective apical traffic pathway. The brush border enzymes DPPIV and sucrase-isomaltase still correctly localize at the apical plasma membrane independent of this pathway. Hence, our work demonstrates how Myo5B, Stx3, Slp4a, Vamp7, Munc18-2, and Rab8/11 cooperate during selective apical cargo trafficking and exocytosis in epithelial cells and thereby provides further insight into MVID pathophysiology.     

Role of dipeptidyl peptidase IV in regulating activity of Na+/H+ exchanger isoform NHE3 in proximal tubule cells

We recently reported that NHE3 exists in multimeric complexes with dipeptidyl peptidase IV (DPPIV) in renal brush-border membranes. To examine the possible role of DPPIV in modulating NHE3 activity, we evaluated whether specific competitive inhibitors that bind to the active site of DPPIV affect NHE3 activity in the OKP line of opossum kidney proximal tubule cells. The DPPIV inhibitors diprotin A and P32/98 significantly reduced NHE3 activity, whereas the inactive isomer P34/98 had no effect. DPPIV inhibitors did not reduce the activity of another brush-border transport process, Na-phosphate cotransport. Effects of DPPIV inhibitors on NHE3 activity were not associated with detectable changes in amount or apparent molecular weight of NHE3 or in NHE3 surface expression. To investigate the signaling mechanisms involved in modulation of NHE3 activity by DPPIV, we used inhibitors of protein kinase pathways known to regulate NHE3. Whereas the PKA inhibitor H-89 failed to block the effect of DPPIV inhibitors, the tyrosine kinase inhibitor genistein alone caused a decrement in NHE3 activity very similar in magnitude to that caused by P32/98. We also found that the effects of genistein and P32/98 on NHE3 activity were not additive. In contrast, forskolin/IBMX and P32/98 had additive inhibitory effects on NHE3 activity. These findings suggested that the effect of DPPIV inhibitors to reduce NHE3 activity results from inhibition of a tyrosine kinase signaling pathway rather than by activation of PKA. We conclude that DPPIV plays an unexpected role in modulating Na⁺/H⁺ exchange mediated by NHE3 in proximal tubule cells. sodium/hydrogen exchange; diprotin A; P32/98; tyrosine kinase     

Acute hypotension induced by aortic clamp vs. PTH provokes distinct proximal tubule Na+ transporter redistribution patterns

Renal parathyroid hormone (PTH) action is often studied at high doses (100 microg PTH/kg) that lower mean arterial pressure significantly, albeit transiently, complicating interpretation of studies. Little is known about the effect of acute hypotension on proximal tubule Na(+) transporters. This study aimed to determine the effects of acute hypotension, induced by aortic clamp or by high-dose PTH (100 microg PTH/kg), on renal hemodynamics and proximal tubule Na/H exchanger isoform 3 (NHE3) and type IIa Na-P(i) cotransporter protein (NaPi2) distribution. Subcellular distribution was analyzed in renal cortical membranes fractionated on sorbitol density gradients. Aortic clamp-induced acute hypotension (from 100 +/- 3 to 78 +/- 2 mmHg) provoked a 62% decrease in urine output and a significant decrease in volume flow from the proximal tubule detected as a 66% decrease in endogenous lithium clearance. There was, however, no significant change in glomerular filtration rate (GFR) or subcellular distribution of NHE3 and NaPi2. In contrast, high-dose PTH rapidly (<2 min) decreased arterial blood pressure to 51 +/- 3 mmHg, decreased urine output, and shifted NHE3 and NaPi2 out of the low-density membranes enriched in apical markers. PTH at much lower doses (<1.4 microg.kg(-1).h(-1)) did not change blood pressure and was diuretic. In conclusion, acute hypotension per se increases proximal tubule Na(+) reabsorption without changing NHE3 or NaPi2 subcellular distribution, indicating that trafficking of transporters to the surface is not the likely mechanism; in comparison, hypotension secondary to high-dose PTH blocks the primary diuretic effect of PTH but does not inhibit the PTH-stimulated redistribution of NHE3 and NaPi2 to the base of the microvilli.