Fluid Volume Homeostasis Research Articles

βPix Up-regulates Na+/H+ Exchanger 3 through a Shank2-mediated Protein-Protein Interaction

Na(+)/H(+) exchanger 3 (NHE3) plays an important role in neutral Na(+) transport in mammalian epithelial cells. The Rho family of small GTPases and the PDZ (PSD-95/discs large/ZO-1) domain-based adaptor Shank2 are known to regulate the membrane expression and activity of NHE3. In this study we examined the role of betaPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2, and betaPix form a macromolecular complex when expressed heterologously in mammalian cells as well as endogenously in rat colon, kidney, and pancreas. In addition, these proteins co-segregated at the apical surface of rat colonic epithelial cells, as detected by immunofluorescence staining. When expressed in PS120/NHE3 cells, betaPix increased membrane expression and basal activity of NHE3. Interestingly, the effects of betaPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor, indicating that Shank2 and Rho GTPases are involved in betaPix-mediated NHE3 regulation. Knockdown of endogenous betaPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that betaPix up-regulates NHE3 membrane expression and activity by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.

Exciting Challenges Ahead For Integrative And Regulatory Physiology

Exciting Challenges Ahead For Integrative And Regulatory Physiology

Mutations in Extracellular Domains Reverse Zn2+ Activation of Human Epithelial Na+ Channels

Epithelial Na+ channels (ENaCs) mediate apical Na+ entry into epithelial cells in kidney, lung and distal colon, playing a critical role in regulation of body fluid volume homeostasis. Several divalent metals modulate ENaC activity in a species-dependent manner. We examined the effect of extracellular Zn2+ on human αβγENaCs expressed in Xenopus oocytes and investigated the underling mechanisms. External Zn2+ increased the whole-cell currents of human ENaCs with a bell-shaped dose response similar to the reported response of mouse ENaC. A peak activation was observed at 100 μM with lower or higher [Zn2+] being less effective. As previously reported for mouse ENaCs, Na+ self-inhibition response of human ENaCs was nearly eliminated in the presence of 100 μM Zn2+, supporting the notion of relieving Na+ self-inhibition as the major mechanism for Zn2+ stimulation of human ENaC currents. We found that mutations of His233 in γ subunit, a residue required for Na+ self-inhibition, converted human ENaC into a Zn2+-inhibited channel with an estimated inhibitory constant of 1 mM. This observation contrasts a previous report of a complete loss of response to Zn2+ in mouse ENaCs bearing homologous mutations. Mutations at two extracellular Cys residues in αENaC but not their homologous residues in γENaC also reversed the stimulatory effect of Zn2+ on human ENaCs. This phenol type cannot be attributed to an absence of Na+ self-inhibition, because the Cys mutants in fact showed enhanced responses to Na+. Our results suggest that activation of human ENaCs by extracellular Zn2+ requires different structural elements from those in mouse ENaCs despite the similar dose response.

Expression of aquaporin 8 is diversely regulated by osmotic stress in amnion epithelial cells

Water absorption across fetal chorioamniotic membranes is a critical regulatory pathway for amniotic fluid volume homeostasis. Aquaporin 8 (AQP8) is a water channel regulating osmotic water travel across membranes. This study was to investigate the distribution and expression of AQP8 in amnion epithelial cells (AEC) in response to different osmotic stresses. Cells derived from the amnion were subjected to different osmotic culture media. Reverse transcriptase-polymerase chain reaction, Western blotting and immunofluorescence analysis were used to determine expression and localization of AQP8. Immunofluorescent staining confirmed the expression of AQP8 on cytomembrane and in cytoplasm. Hypotonic media increased AQP8 on cytomembrane of AEC. Compared to isosmolar media, hypotonic media significantly enhanced AQP8 mRNA and protein expression (P < 0.05), while hypertonic media significantly decreased expression (P < 0.05). The expression and distribution of AQP8 in AEC are diversely regulated by osmotic loads suggesting a role for AQP8 in intramembranous water transport and the balance of amniotic fluid.

Metaplasticity of Hypothalamic Synapses following In Vivo Challenge

Neural networks that regulate an organism's internal environment must sense perturbations, respond appropriately, and then reset. These adaptations should be reflected as changes in the efficacy of the synapses that drive the final output of these homeostatic networks. Here we show that hemorrhage, an in vivo challenge to fluid homeostasis, induces LTD at glutamate synapses onto hypothalamic magnocellular neurosecretory cells (MNCs). LTD requires the activation of postsynaptic alpha2-adrenoceptors and the production of endocannabinoids that act in a retrograde fashion to inhibit glutamate release. In addition, both hemorrhage and noradrenaline downregulate presynaptic group III mGluRs. This loss of mGluR function allows high-frequency activity to potentiate these synapses from their depressed state. These findings demonstrate that noradrenaline controls a form of metaplasticity that may underlie the resetting of homeostatic networks following a successful response to an acute physiological challenge.

Adrenal hormones are necessary for the hydromineral phenotypes of transgenic mice with central renin‐angiotensin system overactivity

To examine the roles of the central renin‐angiotensin system (RAS) in blood pressure and fluid volume homeostasis, our lab previously developed the sRA mouse model, in which human renin is expressed via the neuron‐specific synapsin promoter, and human angiotensinogen via its endogenous promoter. Due to species‐specificity of this reaction, RAS overactivity is restricted to the brain. sRA mice exhibit polydipsia (control, 0.04±0.01 vs sRA, 0.31±0.12 mL/g/d), polyuria (0.08±0.02 vs 0.92±0.04 g/g/d), and a robust sodium appetite (0.94±0.18 vs 3.93±0.07 mEq/d). Here, we examined whether elevated HPA‐axis activity is necessary for these phenotypes. Four weeks after total adrenalectomy, the sodium appetite of sRA mice was completely normalized (0.72±0.21 mEq/d), and the polydipsia (0.11±0.01 mL/g/d) and polyuria (0.15±0.01 g/g/d) were significantly attenuated. In a separate cohort, acute spironolactone treatment partially blocked the sodium intake of sRA mice (3.03±0.31 down to 2.33±0.44 mEq/d), suggesting the involvement of multiple adrenal hormones in this behavior. From these data, we conclude that multiple adrenal hormones are necessary for the dipsogenic and sodium appetite behaviors of mice with chronic overactivity of the brain RAS. Ongoing work is dissecting the contribution of glucocorticoids in hydromineral regulation in sRA mice. (JLG supported by the APS; CLG, AKJ, CDS supported by NIH)

Palm domains of the epithelial sodium channel and sodium self‐inhibition

Epithelial Na+ channels (ENaC) play an essential role in body fluid volume homeostasis by mediating apical Na+ entry in epithelial cells. The structure of chicken acid sensing ion channel 1 (cASIC1) homologous to ENaCs reveals that the palm domain offers the sole link between the extracellular domain and the channel pore within a subunit. We investigated the functional roles of the two charged residues within a palm domain β‐sheet (β12) in the mechanism of Na+ self‐inhibition by site‐directed mutagenesis. The Na+ self‐inhibition responses for wild type and mutant channels expressed in Xenopus oocytes were examined by two electrode voltage clamp. Charge reversal mutations at a homologous site (αK561E and γR520E) of β12 significantly reduced the magnitude and speed of Na+ self‐inhibition response. The Na+ inhibition was absent in oocytes expressing the double mutant channel (αK561E βγR520E). Mutation of the other charged residue in γ subunit (γE524R) also significantly suppressed Na+ self‐inhibition whereas the homologous mutation in α(α E565R) did not alter the inhibition. Our results suggest that certain charged residues in αand γpalm domains are critical determinants for Na+ self‐inhibition. Based on our observations and the insights from the cASIC1 structure, we propose that the palm domains play a pivotal role in Na+ self‐inhibition of ENaC. (Supported by NIH ES14701, DK54354 and DK079307)

Substitutions of a pair of histidine residues within the extracellular loop of alpha subunit of the epithelial sodium channel suppress sodium self‐inhibition

Epithelial Na+ channels (ENaC) mediate apical Na+ transport and play an essential role in body fluid volume homeostasis. Recent studies have demonstrated a key role of the extracellular Na+ concentration in controlling the open probability of ENaC through Na+ self‐inhibition. Here we investigated the role of a pair of adjacent His residues within the extracellular loop in Na+ self‐inhibition. The His325 and His326 of mouse αENaC were mutated to di‐Ala residues. The Na+ self‐inhibition responses for wild type and mutant channels expressed in Xenopus oocytes were examined by two electrode voltage clamp. A significantly suppressed (50%) and slowed (65%) Na+ self‐inhibition was observed in oocytes expressing αH325A‐H326Aβγ, compared to the wild type. Based on the recently published structure of the chicken acid sensing ion channel 1 (cASIC1) and a sequence alignment with αENaC, the di‐His residues are predicted to be located at the C‐terminus of β4 within the β‐ball domain. Interestingly, residue Arg191 of cASIC1, which is adjacent to the sequence homologous to HH of αENaC, directly interacts with a residue (Asp350) within the thumb domain that is predicted to bind proton. Based on our results and the cASIC1 structure, we propose that the di‐His residues interact with the thumb domain and the intrasubunit interaction is involved in the conformational changes induced by the binding of extracellular Na+. (Supported by NIH DK54354)

The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells

We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct ((MPK)CCD(14)) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-beta-cyclodextrin added apically to (MPK)CCD(14) cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 +/- 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of (MPK)CCD(14) cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.

Functional Role of Extracellular Loop Cysteine Residues of the Epithelial Na+ Channel in Na+ Self-inhibition

The epithelial Na(+) channel (ENaC) is typically formed by three homologous subunits (alpha, beta, and gamma) that possess a characteristic large extracellular loop (ECL) containing 16 conserved cysteine (Cys) residues. We investigated the functional role of these Cys residues in Na(+) self-inhibition, an allosteric inhibition of ENaC activity by extracellular Na(+). All 16 Cys residues within alpha and gamma ECLs and selected beta ECL Cys residues were individually mutated to alanine or serine residues. The Na(+) self-inhibition response of wild type and mutant channels expressed in Xenopus oocytes was determined by whole cell voltage clamp. Individual mutation of eight alpha (Cys-1, -4, -5, -6, -7, -10, -13, or -16), one beta (Cys-7), and nine gamma (Cys-3, -4, -6, -7, -10, -11, -12, -13, or -16) residues significantly reduced the magnitude of Na(+) self-inhibition. Na(+) self-inhibition was eliminated by simultaneous mutations of either the last three alpha ECL Cys residues (Cys-14, -15, and -16) or Cys-7 within both alpha and gamma ECLs. By analyzing the Na(+) self-inhibition responses and the effects of a methanethiosulfonate reagent on channel currents in single and double Cys mutants, we identified five Cys pairs within the alphaECL (alphaCys-1/alphaCys-6, alphaCys-4/alphaCys-5, alphaCys-7/alphaCys-16, alphaCys-10/alphaCys-13, and alphaCys-11/alphaCys-12) and one pair within the gammaECL (gammaCys-7/gammaCys-16) that likely form intrasubunit disulfide bonds. We conclude that approximately half of the ECL Cys residues in the alpha and gamma ENaC subunits are required to establish the tertiary structure that ensures a proper Na(+) self-inhibition response, likely by formation of multiple intrasubunit disulfide bonds.

Shank2 Associates with and Regulates Na+/H+ Exchanger 3

Na+/H+ exchanger 3 (NHE3) plays a pivotal role in transepithelial Na+ and HCO3(-) absorption across a wide range of epithelia in the digestive and renal-genitourinary systems. Accumulating evidence suggests that PDZ-based adaptor proteins play an important role in regulating the trafficking and activity of NHE3. A search for NHE3-binding modular proteins using yeast two-hybrid assays led us to the PDZ-based adaptor Shank2. The interaction between Shank2 and NHE3 was further confirmed by immunoprecipitation and surface plasmon resonance studies. When expressed in PS120/NHE3 cells, Shank2 increased the membrane expression and basal activity of NHE3 and attenuated the cAMP-dependent inhibition of NHE3 activity. Furthermore, knock-down of native Shank2 expression in Caco-2 epithelial cells by RNA interference decreased NHE3 protein expression as well as activity but amplified the inhibitory effect of cAMP on NHE3. These results indicate that Shank2 is a novel NHE3 interacting protein that is involved in the fine regulation of transepithelial salt and water transport through affecting NHE3 expression and activity.

Nongenomic regulation by aldosterone of the epithelial NHE3 Na+/H+ exchanger

The relevance of nongenomic pathways to regulation of epithelial function by aldosterone is poorly understood. Recently, we demonstrated that aldosterone inhibits transepithelial HCO(3)(-) absorption in the renal medullary thick ascending limb (MTAL) through a nongenomic pathway. Here, we examined the transport mechanism(s) responsible for this regulation, focusing on Na(+)/H(+) exchangers (NHE). In the MTAL, apical NHE3 mediates H(+) secretion necessary for HCO(3)(-) absorption; basolateral NHE1 influences HCO(3)(-) absorption by regulating apical NHE3 activity. In microperfused rat MTALs, the addition of 1 nM aldosterone rapidly decreased HCO(3)(-) absorption by 30%. This inhibition was unaffected by three maneuvers that inhibit basolateral Na(+)/H(+) exchange and was preserved in MTALs from NHE1 knockout mice, ruling out the involvement of NHE1. In contrast, exposure to aldosterone for 15 min caused a 30% decrease in apical Na(+)/H(+) exchange activity over the intracellular pH range from 6.5 to 7.7, due to a decrease in V(max). Inhibition of HCO(3)(-) absorption by aldosterone was not affected by 0.1 mM lumen Zn(2+) or 1 mM lumen DIDS, arguing against the involvement of an apical H(+) conductance or apical K(+)-HCO(3)(-) cotransport. These results demonstrate that aldosterone inhibits HCO(3)(-) absorption in the MTAL through inhibition of apical NHE3, and identify NHE3 as a target for nongenomic regulation by aldosterone. Aldosterone may influence a broad range of epithelial transport functions important for extracellular fluid volume and acid-base homeostasis through direct regulation of this exchanger.

Natriuretic Peptides and the Dialysis Patient

The natriuretic peptide family consists of four structurally similar, but genetically distinct molecules with pronounced cardiovascular and renal actions. They are counterregulatory hormones playing an important role in fluid volume homeostasis. Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) cause diuresis, natriuresis, and vasodilatation. C-type natriuretic peptide (CNP) has antimitogenic effects and causes vascular smooth muscle relaxation. Dendroaspis natriuretic peptide (DNP) shares many of the actions of ANP and BNP, but its function in humans is not yet fully understood. Natriuretic peptides have been extensively investigated as biochemical markers of the fluid state. Levels are elevated in disease conditions characterized by fluid overload and are closely related to survival in various cardiac disease states. In the dialysis population, BNP correlates significantly with cardiac function, whereas ANP is sensitive to volume changes during dialysis. However, changes in concentration do not predict achievement of euvolemia, and short half-life, combined with complicated assay techniques, make ANP a less than satisfactory tool for assessing hydration. BNP is a superior prognosticator for risk stratification in dialysis patients, and serial estimations will help in the identification of occult cardiac disease.

Biology of natriuretic peptides and their receptors

Increasing evidence suggests that natriuretic peptides (NPs) play diverse roles in mammals, including renal hemodynamics, neuroendocrine, and cardiovascular functions. Collectively, NPs are classified as hypotensive hormones; the main actions of NPs are implicated in eliciting natriuretic, diuretic, steroidogenic, antiproliferative, and vasorelaxant effects, important factors in the control of body fluid volume and blood pressure homeostasis. One of the principal loci involved in the regulatory actions of NPs is their cognate plasma membrane receptor molecules, which are activated by binding with specific NPs. Interaction of NPs with their receptors plays a central role in physiology and pathophysiology of hypertension and cardiovascular disorders. Gaining insight into the intricacies of NPs-specific receptor signaling pathways is of pivotal importance for understanding both hormone-receptor biology and the disease states arising from abnormal hormone receptor interplay. During the last decade there has been a surge in interest in NP receptors; consequently, a wealth of information has emerged concerning molecular structure and function, signaling mechanisms, and use of transgenics and gene-targeted mouse models. The objective of this present review is to summarize and document the previous findings and recent discoveries in the field of the natriuretic peptide hormone family and receptor systems with emphasis on the structure–function relationship, signaling mechanisms, and the physiological and pathophysiological significance in health and disease.

Extracellular Histidine Residues Crucial for Na+ Self-inhibition of Epithelial Na+ Channels

Epithelial Na(+) channels (ENaC) participate in the regulation of extracellular fluid volume homeostasis and blood pressure. Channel activity is regulated by both extracellular and intracellular Na(+). The down-regulation of ENaC activity by external Na(+) is referred to as Na(+) self-inhibition. We investigated the structural determinants of Na(+) self-inhibition by expressing wild-type or mutant ENaCs in Xenopus oocytes and analyzing changes in whole-cell Na(+) currents following a rapid increase of bath Na(+) concentration. Our results indicated that wild-type mouse alphabetagammaENaC has intrinsic Na(+) self-inhibition similar to that reported for human, rat, and Xenopus ENaCs. Mutations at His(239) (gammaH239R, gammaH239D, and gammaH239C) in the extracellular loop of the gammaENaC subunit prevented Na(+) self-inhibition whereas mutations of the corresponding His(282) in alphaENaC (alphaH282D, alphaH282R, alphaH282W, and alphaH282C) significantly enhanced Na(+) self-inhibition. These results suggest that these two histidine residues within the extracellular loops are crucial structural determinants for Na(+) self-inhibition.

Gastric emptying in human fetus: when does it start?

Fetal swallowing and gastric emptying contribute to amniotic fluid volume (AFV) homeostasis and fetal gastrointestinal development. As fetal urine contributes to AFV by the end of the first trimester, we speculated that fetal swallowing and gastric emptying must be functional to prevent rapid increases in AFV. We sought to determine the gestation for initiation of gastric emptying in the human fetus.

Spatiotemporal regulation of the two atrial natriuretic peptide receptors in testis.

By interacting with a guanylyl cyclase (GC) activity-containing receptor, termed GC-A, atrial natriuretic peptide (ANP) acts as a regulator of blood pressure and fluid volume homeostasis. High expression levels of GC-A in the testis and reported effects of ANP on testosterone secretion by Leydig cells are indicative of important local functions in this organ. Here we show, based on radioligand receptor labeling and immunological approaches, that seminiferous tubules rather than Leydig cells are the predominant GC-A expression sites in the rat testis. Functional activity was proved by ANP- induced cGMP accumulation in isolated seminiferous tubules. Although ontogenetic studies revealed a massive increase in GC-A levels during sexual maturation, the so-called natriuretic peptide clearance receptor, another type of ANP receptor proposed to locally control the availability of natriuretic peptides, was found to be expressed predominantly before puberty, exceeding the level of GC-A expression at this time. Natriuretic peptide clearance receptor also shows a distinct distribution pattern surrounding the seminiferous tubules. These findings raise the possibility of novel physiological roles for ANP and cGMP in the testis related to germ cell maturation and/or the regulation of the onset of puberty and suggest that the two ANP receptors function in a coordinated manner at this target organ.

Diversity of the mammalian sodium/proton exchanger SLC9 gene family.

Sodium/proton antiporters or exchangers (NHE) are integral membrane proteins present in most, if not all, living organisms. In mammals, these transporters chiefly catalyze the electroneutral exchange of Na(+) and H(+) down their respective concentration gradients and are crucial for numerous physiological processes, ranging from the fine control of intracellular pH and cell volume to systemic electrolyte, acid-base and fluid volume homeostasis. NHE activity also facilitates the progression of other cellular events such as adhesion, migration, and proliferation. Thus far, eight distinct NHE genes (NHE1/SLC9A1-NHE8/SLC9A8) and several pseudogenes have been identified in the human genome. The functional genes encode proteins of varying primary sequence identity (25-70%), but share a common predicted secondary structure comprising 12 conserved membrane-spanning segments at the amino-terminus and a more divergent, cytoplasmically-oriented, carboxy-terminus. They show considerable heterogeneity in their patterns of tissue/cell expression and membrane localization. Functional studies have revealed further differences in their kinetic properties, sensitivity to pharmacological antagonists, and regulation by diverse hormonal and mechanical stimuli. Altered NHE activity has been linked to the pathogenesis of several diseases, including essential hypertension, congenital secretory diarrhea, diabetes, and tissue damage caused by ischemia/reperfusion. Further characterization of their functional properties should lead to a better understanding of their unique contributions to human health and disease.

Expression of aquaporin 8 and its up-regulation by cyclic adenosine monophosphate in human WISH cells

Objective: Water absorption across the fetal chorioamniotic membranes is a critical regulatory pathway for amniotic fluid volume homeostasis. Aquaporins are cell membrane proteins that significantly enhance membrane permeability to water by acting as water channels. We recently demonstrated that aquaporin 8 is expressed in human amnion, chorion, and placenta. Thus, aquaporin 8 expression represents a molecular mechanism of amniotic water absorption through intramembranous pathways. The current study sought to determine whether aquaporin 8 is expressed in human amnion-derived cell culture and to explore its regulation by second messenger cyclic adenosine monophosphate. Study Design: Human amnion–derived WISH cells were cultured. Total RNA was isolated and reverse transcriptase-polymerase chain reaction was used to determine aquaporin 8 gene expression. To determine the effect of cyclic adenosine monophosphate on aquaporin 8 expression, WISH cells were cultured in the presence of either monobutyryl cyclic adenosine monophosphate or the cyclic adenosine monophosphate–elevating agent forskolin. Multiplex semiquantitative reverse transcriptase–polymerase chain reaction was carried out to quantify aquaporin 8 messenger RNA levels. Results: Reverse transcriptase–polymerase chain reaction detected aquaporin 8 expression in WISH cells. After forskolin treatment for 2 hours, aquaporin 8 messenger RNA expression in WISH cells increased 4-fold (P <.001). Stimulation of aquaporin 8 gene expression by colforsin was observed throughout the study period of 20 hours. Incubation of WISH cells with monobutyryl cyclic adenosine monophosphate resulted in a 2-fold increase in aquaporin 8 messenger RNA level (P <.001). However, stimulation of aquaporin 8 gene expression by monobutyryl cyclic adenosine monophosphate attenuated to baseline level after 20 hours of monobutyryl cyclic adenosine monophosphate treatment. Conclusion: The current study demonstrates the expression of aquaporin 8 water channel in human amnion–derived WISH cells and aquaporin 8 expression up-regulation by second messenger cyclic adenosine monophosphate. Aquaporin 8 messenger RNA demonstrates a relatively short biologic half-life in vitro, which renders its rapid responsiveness to regulation (Am J Obstet Gynecol 2003;188:997-1001.)

Renal function in NHE3-deficient mice with transgenic rescue of small intestinal absorptive defect.

The degree to which loss of the NHE3 Na(+)/H(+) exchanger in the kidney contributes to impaired Na(+)-fluid volume homeostasis in NHE3-deficient (Nhe3(-/-)) mice is unclear because of the coexisting intestinal absorptive defect. To more accurately assess the renal effects of NHE3 ablation, we developed a mouse with transgenic expression of rat NHE3 in the intestine and crossed it with Nhe3(-/-) mice. Transgenic Nhe3(-/-) (tgNhe3(-/-)) mice tolerated dietary NaCl depletion better than nontransgenic knockouts and showed no evidence of renal salt wasting. Unlike nontransgenic Nhe3(-/-) mice, tgNhe3(-/-) mice tolerated a 5% NaCl diet. When fed a 5% NaCl diet, tgNhe3(-/-) mice had lower serum aldosterone than tgNhe3(-/-) mice on a 1% NaCl diet, indicating improved extracellular fluid volume status. Na(+)-loaded tgNhe3(-/-) mice had sharply increased urinary Na(+) excretion, reflective of increased absorption of Na(+) in the small intestine; nevertheless, they remained hypotensive, and renal studies showed a reduction in glomerular filtration rate (GFR) similar to that observed in nontransgenic Nhe3(-/-) mice. These data show that reduced GFR, rather than being secondary to systemic hypovolemia, is a major renal compensatory mechanism for the loss of NHE3 and indicate that loss of NHE3 in the kidney alters the set point for Na(+)-fluid volume homeostasis.