Renal function in NHE3-deficient mice with transgenic rescueof small intestinal - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:37 |只看该作者 |倒序浏览 |打印

作者：Alison L.Woo, William T.Noonan, Patrick J.Schultheis, Jonathan C.Neumann, Patrice A.Manning, John N.Lorenz,  Gary E.Shull作者单位：Departments of Molecular Genetics, Biochemistry,and Microbiology and Molecular andCellular Physiology, The University of Cincinnati College ofMedicine, Cincinnati, Ohio 45267-0524;and Department of Biological Sciences, NorthernKentucky University, Highland Heights, Kentucky 41099



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      【摘要】. ]/ \2 W) K# C8 V
   The degree to which loss of theNHE3 Na /H exchanger in the kidney contributesto impaired Na -fluid volume homeostasis in NHE3-deficient( Nhe3 / ) mice is unclear because of thecoexisting intestinal absorptive defect. To more accurately assess therenal effects of NHE3 ablation, we developed a mouse with transgenicexpression of rat NHE3 in the intestine and crossed it with Nhe3 / mice. Transgenic Nhe3 / (tg Nhe3 / )mice tolerated dietary NaCl depletion better than nontransgenic knockouts and showed no evidence of renal salt wasting. Unlike nontransgenic Nhe3 / mice,tg Nhe3 / mice tolerated a 5% NaCl diet. Whenfed a 5% NaCl diet, tg Nhe3 / mice had lowerserum aldosterone than tg Nhe3 / mice on a 1%NaCl diet, indicating improved extracellular fluid volume status.Na -loaded tg Nhe3 / mice hadsharply increased urinary Na excretion, reflective ofincreased absorption of Na in the small intestine;nevertheless, they remained hypotensive, and renal studies showed areduction in glomerular filtration rate (GFR) similar to that observedin nontransgenic Nhe3 / mice. These data showthat reduced GFR, rather than being secondary to systemic hypovolemia,is a major renal compensatory mechanism for the loss of NHE3 andindicate that loss of NHE3 in the kidney alters the set point forNa -fluid volume homeostasis.
      【关键词】 sodium/hydrogen exchanger diarrhea Slca sodium absorption sodiumfluid volume homeostasis* \; a% |1 s% x4 t0 r
   INTRODUCTION

N A /H exchanger isoform 3 (NHE3) is one of several Na transport proteins in renal epithelial cells that are involved inmaintaining Na -fluid volume homeostasis. Localized toapical membranes of the proximal tubule and to a lesser extent in thethick ascending limb of Henle, NHE3 transports Na into thecell in exchange for H and is responsible for absorbinglarge quantities of NaCl and HCO 3 −, with accompanyingwater ( 1, 3, 24 ). NHE3 null mutant( Nhe3 / ) mice have severe absorptive defectsin both the kidney and intestine, and they exhibit characteristics ofchronic volume depletion, including low blood pressure, high levels ofrenin mRNA in kidney, and high serum aldosterone ( 21 ). Insitu microperfusion and micropuncture studies showed that reabsorptionof HCO 3 − and water was reduced in the proximal tubuleof Nhe3 / mice ( 12, 24 ).However, fluid delivery to the distal convoluted tubule was notsignificantly different from that in wild-type mice, and this appearedto be due to a regulated reduction in the glomerular filtration rate(GFR) resulting from tubuloglomerular feedback (TGF) mechanisms( 12 ). These observations suggested that the reduction inGFR might be a compensatory mechanism by which the kidneys of Nhe3 / mice conserve Na andHCO 3 − ( 4, 8, 12 ).0 L* T/ p( q+ o) S  F9 t
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The observed normalization of fluid delivery to the distal convolutedtubule of NHE3-deficient mice ( 12 ) raised the possibility that the proximal tubule absorptive defect itself might not lead tosignificant renal salt wasting. In a subsequent study, when subjectedto dietary Na restriction, Nhe3 / mice lost weight rapidly and did infact exhibit urinary salt wasting ( 8 ), although apparentlynot as severe as in mice lacking transporters along some of the moredistal segments of the nephron, such as the ROMK K channel( 10 ) and Na -K -2Cl cotransporter ( 23 ) of the thick ascending limb and theepithelial Na channel (ENaC) of the connecting tubule andcollecting duct ( 2, 6, 14, 18, 23 ). By the third day ofdietary Na restriction, however, many of the Nhe3 / mice were undergoing hypovolemic renalfailure. This raises the possibility that systemic volume depletion,and not just the loss of NHE3 in the kidney, might cause some degree ofrenal dysfunction. Thus extracellular fluid volume depletion itself,which is exacerbated by the diarrheal state during dietaryNa restriction, may have contributed to the mildimpairment in the ability of the NHE3-deficient kidney to retainNa  . Similarly, it was unclear whether the observedreduction in GFR in Nhe3 / mice might havebeen due, in part, to systemic hypovolemia and hypotension rather thanto an appropriate regulation of fluid delivery to the distal tubule viaTGF mechanisms. Attempts to improve the fluid volume status of Nhe3 / mice by feeding them a high-NaCl dietresulted in swelling of the intestine, severe hypovolemia, and death,further suggesting that the intestinal defect impaired extracellularfluid-volume homeostasis.2 F" j) Y# Y  `8 }6 `( o

Thus the coexisting intestinal absorptive defect and chronic diarrheain Nhe3 / mice represent a major confoundingfactor in determining the specific effects of the loss of NHE3 in thekidney on renal Na conservation, GFR, and extracellularfluid-volume homeostasis. To assess these issues, we developed atransgenic mouse in which NHE3 is expressed in the small intestine viathe intestinal fatty acid binding protein (IFABP) promoter andcrossed it with Nhe3 / mice. Transgenic Nhe3 / (tg Nhe3 / )mice were then subjected to dietary Na restriction andNa loading, and renal function was analyzed. Both dietaryNa restriction and Na loading were bettertolerated in tg Nhe3 / mice than innontransgenic Nhe3 / mice. Salt loading ledto a substantial reduction of aldosterone levels intg Nhe3 / mice, indicating a partialcorrection of the extracellular fluid-volume deficit. However,tg Nhe3 / mice remained mildly hypotensive andhad reduced GFR compared with Nhe3  /  mice alsoharboring the IFABP- Nhe3 transgene(tg Nhe3  /  ).- }4 a5 L8 V' F1 h, r; C, a' y1 r
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METHODS
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Production and genotyping of mutant and transgenic mice. The rat NHE3 cDNA was cloned into an expression plasmid containing thesmall intestine-specific IFABP promoter (nucleotides 1178 to  28) anda t-intron polyadenylation cassette [provided by J. A. Whitsett( 27 )] (Fig. 1 A ).The IFABP/NHE3 construct was microinjected into fertilized oocytes fromInstitute of Cancer Research (ICR) mice, and injected oocytes wereimplanted into the uterus of pseudopregnant mice to produce transgenicanimals by the University of Cincinnati transgenic core facility. Mice carrying the transgene integrated into their genome were identified byPCR analysis. The 5'-oligonucleotide primer sequence was from the IFABPpromoter sequence (5'-CTGCCAGGTTATCTCTTGAAC-3'), and the 3' reverseprimer sequence was from the NHE3 cDNA sequence (5'-CTGTTCGGTTCCTCCTCAATG-3'). PCR conditions were 94°C for 3 min,then 35 cycles at 94°C for 30 s, 60°C for 30 s, and72°C for 30 s, followed by 72°C for 10 min. ICR transgenicmice were backcrossed for two to three generations with Nhe3  / mice of a mixed 129SvJ and Black Swissbackground ( 21 ) to produce Nhe3  /  and Nhe3 / mice carrying the IFABP/NHE3transgene (tg Nhe3  /  andtg Nhe3 / ). Thus the genetic background of themice used in these experiments was 12.5-25% ICR, with theremainder being an equal mix of 129SVJ and Black Swiss. Nhe3  / mice were generated and maintained aspreviously described ( 21 ). PCR genotyping was performedusing the following primers: a forward primer corresponding to asequence in exon 6 (5'-CTTTTGCGGCATCTGCTGTCAG-3'), a reverse primercorresponding to a sequence in intron 6 (5'-ACTACTAAGAGTGCTCCTAGCTCTCACC-3'), and a reverse primercorresponding to a sequence in the neomycin resistance gene(5'-GCATGCTCCAGACTGCCTTG-3'). PCR conditions were 94°C for 3 min,then 40 cycles at 94°C for 30 s, 62°C for 30 s, and72°C for 30 s. The experiments described below were performed inaccordance with the guidelines established by the Institutional AnimalCare and Use Committee at the University of Cincinnati College ofMedicine. All experimental pairs of tg Nhe3 / and tg Nhe3  /  mice were 8-12 wk old andwere littermates matched by both age and sex to ensure that no strain,age, or sex biases contributed to physiological outputs.$ D' M$ c2 {! S* l" H, P

Fig. 1. Transgenic expression of Na /H exchanger isoform 3 (NHE3) mRNA and protein in small intestine. A : diagram of the transgene using the intestinal fatty acidbinding protein (IFABP) promoter to drive expression of rat NHE3 cDNAin small intestine. Arrows represent PCR primers used to identify micewith genomic insertion of the transgene. B : PCR genotypingof transgenic mice showing presence or absence of a 310-bp product intransgenic ( ) or nontransgenic ( ) mice, respectively.H 2 O designates negative control with no DNA added. C : Northern blot analysis of NHE3 mRNA in transgenic Nhe3  /  kidney (K), small intestine (SI), cecum(Ce), and colon (Co); each lane contains 10 µg of RNA. The endogenousNHE3 mRNA is 5.6 kb, the transgene mRNA is 3.5 kb. D :Western blot analysis of NHE3 in small intestine of transgenic andnontransgenic Nhe3  /  and Nhe3 / mice, using 20 µg of total membranesand a rat anti-NHE3 antibody.6 o2 e1 }) V- c: M8 w- }( m1 ]

RNA isolation and Northern blot analysis. Total RNA was extracted from tissues using Tri-Reagent (MolecularResearch Center). Total RNA (10 µg/sample) was mixed with GlyoxalSample Buffer (BioWhittaker Molecular Applications, Rockland, ME),separated by electrophoresis in 1% agarose, and transferred toHybond-N  nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ).Northern blots were screened using [ 32 P]-labeled cDNAprobes specific for NHE3, renin, and the L32 ribosomal protein.Quantitation of renin mRNA levels was determined by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software(Molecular Dynamics). Renin expression levels were normalized using L32ribosomal protein mRNA as a loading control and reported as mean volumeintegrated values for four pairs of tg Nhe3 / and tg Nhe3  /  mice on 1 or 5% NaCl diets.
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Total membrane preparations and Western blot analysis. Small intestines and kidneys were homogenized in homogenization buffer(0.25 M sucrose, 30 mM imidizole, 1 mM EDTA) to which a proteaseinhibitor cocktail without metal chelating reagents (Sigma, St. Louis,MO) was added. Homogenized suspensions were centrifuged at 6,000 g for 15 min at 4°C. The supernatant was saved, and thepellet was homogenized and centrifuged again. The combined supernatantswere centrifuged at 200,000 g for 1 h at 4°C, and thepellet containing total membranes was resuspended in homogenizationbuffer. A BCA protein assay kit (Pierce, Rockford, IL) was used toquantitate protein concentrations. Total membrane preparations wereanalyzed by Western blot analyses using 1 µg/ml of the rabbit NHE3polyclonal antibody (Chemicon, Temecula, CA), as described previously( 26 ). Signal detection was accomplished using theSuperSignal West Pico Chemiluminescent Substrate (Pierce).

Analysis of size and contents of intestinal segments. Mice were anesthetized with intraperitoneal injections of 2.5% avertin(0.02 ml/g body wt) and euthanized by cervical dislocation. Thecontents of each segment were removed, and the weights of the tissueand contents were recorded. Contents were mixed with 1 ml sterilesaline and centrifuged, and the pH of the supernatant was recorded.' q; E& @; n, |2 Z$ L: ?
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Balance studies using varyingNa -content diets. Mice were housed in metabolic cages and provided with drinking waterand food ad libitum, as described previously ( 22 ). Food(Harlan Teklad, Madison, WI) contained normal (1%), low (0.01%), orhigh (5%) levels of NaCl. Body weights and the amount of food andwater consumed were recorded every day. Urine samples were collecteddaily and were processed and analyzed for Na andK concentrations as described previously ( 8 ).3 ]% Y' S  [0 q0 ]0 j5 j
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Serum aldosterone levels. Mice were anesthetized, and blood was drawn by cardiac puncture. Serumwas separated from blood cells and stored at 20°C. Serum sampleswere diluted 1:4 in sterile PBS, and aldosterone concentrations weredetermined using an RIA kit (Diagnostic Products, Los Angeles, CA).! p0 _0 B9 V' w% @# s

Renal hemodynamic measurements. Baseline renal function was determined in two groups of seven pairs oftg Nhe3  /  and tg Nhe3 / mice maintained on either a 1 or a 5% NaCl diet for 5 days. Mice wereanesthetized with ketamine (50 µg/g body wt) and inactin (100 µg/gbody wt) and surgically instrumented for renal measurements asdescribed previously ( 12 ). Immediately after surgery, abolus (3 µl/g body wt) of 1% FITC-inulin and 3% PAH in isotonicsaline was administered. This was followed by a maintenance infusion ofthe same solution at 0.15 µl · min 1 · gbody wt 1. After a 30-min equilibration period, baselinerenal function was determined through two 30-min urine samplescollected through a catheter in the bladder ( 12 ). At themidpoint of each baseline collection, an arterial blood sample (60 µl) was obtained for determination of plasma FITC-inulin( 11 ) and PAH ( 25 ) concentrations, and donorblood was administered to replace the lost volume after each sample wasobtained. At the end of the second baseline collection, another bloodsample was acquired and plasma electrolyte levels were measured using apH/blood-gas analyzer (Bayer, Medfield, MA). Urinary Na and K concentrations were determined using a Corning 480 Flame Photometer (Bayer). GFR was calculated from inulin clearance, andeffective renal plasma flow (ERPF) was calculated from PAH clearance.$ d( O) y: j0 W+ k/ Q) s
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Statistics. Statistical analysis was performed by either Student's t -test or analysis of variance. When analysis of variancewas applied, either a single-factor design or a mixed-factorial designwith repeated measures on the second factor was used, and individual contrasts were used to compare individual group means when needed. Dataare presented as means ± SE, and statistical significance wasregarded as P- O$ Z' V/ x1 ^' C1 V
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RESULTS2 V0 P8 t; j6 |7 W' d6 }8 S

Generation of transgenic mice expressing NHE3 in the smallintestine. No promoters are available that would allow transgenic expression ofNHE3 throughout the intestinal tract. To partially rescue theintestinal absorptive defect of Nhe3 / mice,we used the rat IFABP promoter to drive expression of a rat NHE3transgene (Fig. 1 A ) in the small intestine. Genomicintegration of the transgene was detected by PCR analysis (Fig. 1 B ). Northern blot analysis of kidney, small intestine,cecum, and colon tissue from tg Nhe3  /  micerevealed expression of a 3.5-kb mRNA corresponding to the transgeneonly in small intestine, whereas the 5.6-kb mouse NHE3 mRNA wasexpressed in all tissues (Fig. 1 C ). The transgenic mice werethen bred with Nhe3  / mice. Western blotanalysis demonstrated that NHE3 protein was expressed in kidneys (datanot shown) and small intestines (Fig. 1 D ) of both transgenicand nontransgenic wild-type mice but not in the kidneys (data notshown) of tg Nhe3 / mice. Intg Nhe3 / mice, NHE3 protein was expressed inthe small intestine, although at a lower level than in wild-typecontrols (Fig. 1 D ). In addition to a protein correspondingto full-length rat NHE3, a smaller band of unknown identity was alsodetected in tg Nhe3 / small intestine.

Analysis of intestinal weight and intestinal contents. The loss of NHE3 in the intestinal tract causes a severe absorptivedefect, resulting in chronic diarrhea. All segments of the intestineare enlarged in Nhe3 / mice, and the volumeand pH of the luminal contents are increased ( 21 ).Expression of functional NHE3 in the Nhe3 / small intestine (Fig. 1, C and D ) would beexpected to at least partially alleviate these defects in this segment.Gross examination of the tg Nhe3 / intestinaltract revealed a less bloated small intestine, whereas the cecum andcolon appeared similar to those of Nhe3 / mice ( 21 ). The weight of the small intestine was greaterin tg Nhe3 / mice than intg Nhe3  /  mice; however, the weight and pH ofthe small intestinal contents in tg Nhe3 / mice were not significantly different from those intg Nhe3  /  mice (Fig. 2 ). On the other hand, the weights andluminal contents of the tg Nhe3 / cecum andcolon, where the transgene was not expressed, were significantlygreater than in tg Nhe3  /  mice, and the pH ofthe luminal contents was significantly more alkaline (Fig. 2 ). Thesedata suggest that the observed levels of expression of the NHE3transgene partially restored the absorptive capabilities of the smallintestine.

Fig. 2. Analysis of intestinal segments and contents oftransgenic (tg) Nhe3  /  andtg Nhe3 / mice. A : weight of smallintestine, cecum, and colon after removal of contents. B :weight of luminal contents of small intestine, cecum, and colon. C : pH of luminal contents of small intestine, cecum, andcolon. Values are means ± SE; n = 6/genotype.* P Nhe3  /  values.
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tgNhe3 / mice exhibit increased tolerance for aNa -restricted diet. When Nhe3 / mice not carrying the transgenewere fed a Na -restricted (0.01% NaCl) diet, theyexhibited severe weight loss and renal salt wasting ( 8 ).To test the Na -handling capabilities oftg Nhe3 / mice,tg Nhe3  /  and tg Nhe3 / mice were housed in metabolic cages and fed a 1% NaCl diet for 3 days,followed by a 0.01% NaCl diet for 3 days. On the normal diet (1%NaCl), tg Nhe3 / mice had lower urinaryNa excretion compared withtg Nhe3  /  mice, consistent with only a partialrescue of the intestinal phenotype. However, during dietaryNa restriction, tg Nhe3 / micelost only 6% of their body weight (Table 1 ), which was a major improvement overthe 17% average loss of body weight for Nhe3 / mice subjected to the same protocol( 8 ). Furthermore, in contrast to nonrescued Nhe3 / mice that continued to excretesubstantial amounts of Na even after 3 days on low salt( 8 ), tg Nhe3 / mice lowered theirNa excretion after 3 days to very low levels that were notsignificantly different from that for tg Nhe3  /  mice (Fig. 3 A, Table 1 ).
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Table 1. Balance studies intgNhe3  /  and tgNhe3 / mice on a 0.01% NaCl diet

Fig. 3. Urinary Na excretion bytg Nhe3  /  and tg Nhe3 / mice in response to dietary Na restriction ( A )or dietary Na loading ( B ). A : 5 pairs of mice were fed a 1% NaCl diet on days 1-3 (D1-D3) and then a 0.01% NaCl diet on days 4-6 (D4-D6). B : 5 pairs of mice were fed a 1% NaCl diet on days 1-3 (D1-D3) and then a 5% NaCl diet on days 4-7 (D4-D7). Values are means ± SE.* P Nhe3  /  values.

Some of the metabolic characteristics oftg Nhe3 / mice during dietary Na restriction were similar to those reported for nonrescued Nhe3 / mice ( 8 ). Compared withtg Nhe3  /  controls,tg Nhe3 / mice drank significantly more waterregardless of diet, and they had a greater urinary output when fed a0.01% NaCl diet (Table 1 ). Urinary K excretion was lowerin tg Nhe3 / mice, although the difference wasnot statistically significant and was likely related to increasedintestinal K secretion as a result of the diarrheal state.Consistent recovery of fecal samples fromtg Nhe3 / mice was not possible due to thepersistent diarrhea resulting from the absorptive defect in the cecumand colon.

tgNhe3 / mice fed a 5% NaCl diet have increased urinaryNa excretion indicative of increasedintestinal NaCl absorption. NaCl loading via continuous infusion of saline through a venouscatheter can restore extracellular fluid volume in Nhe3 / mice, as shown by increases in bloodpressure to a level similar to that for Nhe3  /  mice (Lorenz JN, unpublishedobservations). This suggested that it might be possible torestore extracellular fluid volume by dietary NaCl loading. However,when Nhe3 / mice not carrying the transgenewere fed a high-salt diet (5% NaCl), their small intestines becameseverely swollen, probably due to an osmotic effect from the highlevels of NaCl in the gut, and they died within 48 h (Lorenz JNand Shull GE, unpublished observations). In contrast, whentg Nhe3 / mice were fed a normal-salt diet(1% NaCl) for 3 days followed by a 5% NaCl diet for 4 days, they notonly tolerated the high-salt diet, but their urinary Na excretion increased to levels higher than that seen intg Nhe3  /  mice maintained on a normal diet (Fig. 3 B, Table 2 ). These data demonstrate, importantly, that the intestinally rescuedNHE3-deficient mice can compensate for possible urinary NaCl lossesthrough increases in intestinal NaCl absorption, whereas theirnonrescued counterparts could not. There was no difference in weightbetween tg Nhe3 / andtg Nhe3  /  mice fed either diet, although bodyweight decreased slightly in both genotypes when on the 5% NaCl diet(Table 2 ). Again, tg Nhe3 / mice drank morewater than tg Nhe3  /  mice regardless of diet,and both genotypes consumed more water and increased their urinaryoutput when fed the 5% NaCl diet (Table 2 ). Urinary K excretion was not different between the two genotypes fed either diet(Table 2 ). These data indicate that dietary salt loading increasedintestinal Na absorption intg Nhe3 / mice.

Table 2. Balance studies intgNhe3  /  and tgNhe3 / mice on a 5% NaCl diet: ]- n5 k6 U- @

Serum aldosterone and renin mRNA levels in the kidney are decreasedintgNhe3 / mice fed a 5% NaCl diet. The major hormonal mechanism for correction of a deficit inextracellular fluid volume is an increase in serum aldosterone, whichstimulates the absorption of NaCl in the kidney and intestine. When feda diet containing 1% NaCl, tg Nhe3 / mice hada serum aldosterone level that was similar to that observed previouslyin Nhe3 / mice ( 21 ) and was11-fold greater than that in tg Nhe3  /  mice(Fig. 4 ). These data indicate thattg Nhe3 / mice fed a 1% NaCl diet have asevere deficit in extracellular fluid volume. Serum aldosteronewas sharply decreased in tg Nhe3 / mice whenthey were fed a 5% NaCl diet, although it was still higher than thatin tg Nhe3  /  mice (Fig. 4 ), indicating that thedeficit in extracellular fluid volume can be partially corrected bydietary salt loading.
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Fig. 4. Serum aldosterone levels intg Nhe3  /  and tg Nhe3 / mice. Serum samples were taken from mice maintained on either a 1%NaCl ( n = 6/genotype) or a 5% NaCl ( n = 4/genotype) diet. Values are means ± SE. * P Nhe3  /  values. P, e$ }9 }+ V* ~7 A' f  b8 ~
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Volume depletion also activates the intrarenal renin-angiotensinsystem, as indicated by an increase in the expression of renin mRNA inthe kidney in response to dietary Na restriction( 7 ). As shown in Fig. 5,renin mRNA levels were about sevenfold greater intg Nhe3 / kidneys than intg Nhe3  /  kidneys when the mice were fed a 1%NaCl diet but were only about twofold greater when they were fed a 5%NaCl diet. The reduced level of renin mRNA induction suggests that thevolume status of tg Nhe3 / mice was improvedby dietary salt loading.

Fig. 5. Northern blot analysis of renin mRNA in kidneys fromtg Nhe3  /  and tg Nhe3 / mice. A : blots containing RNA samples (10 µg/lane) fromindividual kidneys from 4 pairs of mice on a 1% NaCl diet and 4 pairsof mice on a 5% NaCl diet were hybridized with a renin probe and thenstripped and hybridized with a probe for the ribosomal L32 protein(loading control). B : renin mRNA hybridization signals werequantitated by PhosphorImager analysis and normalized to the signal forL32 mRNA in that sample. Fold-changes intg Nhe3 / samples relative totg Nhe3  /  samples are represented as means ± SE. * P
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Renal function intgNhe3 / mice fed a 1 or 5% NaCl diet. After 5 days on either a 1 or a 5% NaCl diet,tg Nhe3  /  and tg Nhe3 / mice were anesthetized and surgically prepared for analysis of renalfunction, blood pressure and heart rate, and collection of bloodsamples. Under anesthesia, mean arterial pressure (Fig. 6 A ) was lower intg Nhe3 / mice than intg Nhe3  /  mice regardless of diet, butadministration of the high-salt diet increased blood pressure intg Nhe3 / mice (88.4 ± 3.2 compared with77.0 ± 4.6 mmHg when fed the 1% NaCl diet), whereas it had noeffect in tg Nhe3  /  mice. There were nosignificant differences in heart rate between any of the groups (Fig. 6 B ). Also, in tg Nhe3  /  mice, thehematocrit did not change when animals were placed on the high-saltdiet, but in tg Nhe3 / animals, the 5% NaCldiet significantly reduced the hematocrit (Table 3 ). The effects of high-salt intake onblood pressure and hematocrit are consistent with a partial correctionof the extracellular fluid volume deficit in NHE3-deficient miceexpressing the NHE3 transgene in the small intestine. None of thegroups differed with respect to plasma Na or arterialblood HCO 3 −, but blood pH was significantly lower intg Nhe3 / mice regardless of diet (Table 3 ).Plasma K increased significantly intg Nhe3  /  mice on the 5% NaCl diet comparedwith the same genotype on the 1% NaCl diet, but this increase did notoccur in tg Nhe3 / mice (Table 3 ).2 T' h- ]+ K: \: q# s$ l

Fig. 6. Mean arterial pressure ( A ) and heart rate( B ) of tg Nhe3  /  andtg Nhe3 / mice fed either a 1 or a 5% NaCldiet. Groups of 7 experimental pairs were fed each of the diets for 5 days, anesthetized, and surgically instrumented for analysis ofcardiovascular parameters (shown here), blood, and renal function(shown in Table 3 ). * P Nhe3  /  values.
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Table 3. Blood and renal measurements intgNhe3  /  and tgNhe3 / mice on a 1 or 5% NaCl diet
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GFR and ERPF were lower in tg Nhe3 / mice thanin tg Nhe3  /  mice, and the effect wasindependent of diet (Table 3 ). Furthermore, administration of ahigh-salt diet did not alter GFR in either group. Renal plasma flow, onthe other hand, increased in response to the high-salt diet intg Nhe3  /  animals but not intg Nhe3 / animals. Urinary flow rate was notdifferent between the groups and increased comparably in both groups ofmice in response to high-NaCl intake. The amounts of filteredNa and K were significantly less intg Nhe3 / mice on either diet, consistent withthe reduction in GFR, but the filtered load did not change in responseto a high-NaCl diet in either group. Na excretion andfractional Na excretion were both lower intg Nhe3 / mice than intg Nhe3  /  mice, regardless of diet (Table 3 ),and the sodium excretory response to a high-salt diet was the same inboth genotypes. There were no significant differences in K excretion among any of the groups, but fractional K excretion was significantly reduced in tg Nhe3  /  mice on the 5% NaCl diet compared withtg Nhe3  /  mice on the 1% NaCl diet, consistentwith increased plasma K  .

DISCUSSION

Previous studies demonstrated that both Na reabsorption in the proximal tubule and systemic Na -fluidvolume homeostasis are severely perturbed in Nhe3 / mice ( 8, 12, 21, 24 ) andthat partial compensation for the renal absorptive defect occurs by areduction in GFR ( 4, 8, 12 ). However, Nhe3 / mice also have an intestinalabsorptive defect, which clearly contributes to volume depletion duringdietary Na restriction and might also play a role involume depletion under normal conditions. This makes it difficult toaccurately assess 1 ) the capacity of the NHE3-deficientkidney to recover NaCl; 2 ) the degree to which the reductionin GFR represents direct compensation for the proximal tubuleabsorptive defect, via renal mechanisms such as TGF ( 12 )and the intrarenal renin-angiotensin system ( 15, 19 ); and 3 ) the specific contribution of the renal defect to chronicextracellular fluid volume depletion.

To resolve these issues, we generated Nhe3 / mice expressing NHE3 in the small intestine with the expectation thatthis would allow dietary salt loading, an approach that was notsuccessful with nontransgenic Nhe3 / mice,apparently because of the osmotic effects of high salt in the lumen ofthe gut. It would have been preferable to express NHE3 in smallintestine, cecum, and colon; however, no promoters were available thatwould allow this. Transgenic expression of rat NHE3 under the controlof the IFABP promoter yielded lower levels of expression than that ofthe endogenous protein, and expression was limited to the smallintestine and therefore did not correct the absorptive defect in thececum or colon. In addition to an NHE3 protein corresponding in size towild-type NHE3, there was a diffuse product of lower molecular weightin the tg Nhe3 / small intestine; the identityof this smaller product is unclear. It is possible that glycosylationor trafficking of NHE3, expressed from the transgene, is inefficient inthe Golgi complex of mouse small intestinal epithelial cells. Analternative possibility is that the rat NHE3 mRNA derived from thetransgene, which lacks most of the wild-type 5'- and 3'-untranslatedsequences, may not be efficiently localized to endoplasmicreticulum-bound ribosomes, as there is evidence that untranslated mRNAsequences might be important in this process ( 16 ). Eitherof these possibilities might explain the apparently smaller orpartially degraded NHE3 and the discrepancy between the amount of mRNAexpressed from the transgene and the relatively low amount of normalNHE3 protein detected. Nevertheless, these low levels of NHE3expression did lead to normalization of the pH of small intestinalcontents and, as discussed below, allowed a substantial amount of saltloading when the mice were fed a 5% NaCl diet, thereby eliminating the diarrheal state as a major factor in extracellular fluid volume depletion.) f- G( x  `- \

When the mice were maintained on a 1% NaCl diet, transgenic expressionof NHE3 in the small intestine of Nhe3 / micedid not appear to improve extracellular fluid volume status, as theyhad elevated levels of serum aldosterone, highly induced renin mRNA inkidney, and low blood pressure similar to that seen in nontransgenic Nhe3 / mice ( 21 ). The netintestinal absorption and urinary excretion of NaCl (which must beequivalent when the mice are in balance) corresponded to ~0.22 and~2.1 ml of isotonic fluid/day for tg Nhe3 / and tg Nhe3  /  mice, respectively, consistentwith the possibility that poor absorption of NaCl from the intestinaltract was a major factor in systemic volume depletion. Surprisingly, inresponse to dietary Na restriction,tg Nhe3 / mice exhibited little evidence ofurinary salt wasting. The relatively small reduction in body weight,relative to that observed earlier for Na -restricted Nhe3 / mice ( 8 ), is probably dueprimarily to intestinal losses of NaCl because urinary losses on days 2 and 3 of Na restriction wereonly equivalent to ~0.015 ml of isotonic fluid/day. These resultsindicate that the NHE3-deficient kidney has a substantial ability torecover NaCl and suggest that the hypovolemic renal failure observed inour previous study after 3 days of dietary Na restriction( 8 ), which was a likely factor in the observed renal saltwasting, was brought on largely by the continuing intestinal losses ofsalt and water.6 x/ A3 [) z6 I4 C: L

When the mice were maintained on a 5% NaCl diet, urinaryNa excretion in tg Nhe3 / miceincreased to a level ~18 times that intg Nhe3 / mice on a normal 1% NaCl diet and~2.5 times that in tg Nhe3  /  mice on a 1%NaCl diet. The net urinary excretion (and intestinal absorption) ofNaCl per day in tg Nhe3 / mice was equivalentto that in ~4.3 ml of extracellular fluid and was far in excess ofthat excreted by the NHE3-deficient kidney when the mice were fedeither a normal or a Na -restricted diet (Tables 1 and 2 ).This indicates that the effects of the intestinal absorptive defect onextracellular fluid volume homeostasis can be overcome by dietary saltloading in these animals. As shown by the sharply reduced serumaldosterone levels, the decrease in the level of induction of reninmRNA in the kidney, and the increase in mean arterial pressure, theextracellular fluid volume status of tg Nhe3 / mice was substantially improved when they were fed the 5% NaCl diet.Nevertheless, serum aldosterone and kidney renin mRNA were stillelevated and blood pressure was still reduced relative to that fortg Nhe3  /  mice. These data suggest that theabsence of NHE3 in the kidney, even when the mice are subjected todietary NaCl loading, results in a certain degree of chronic volumedepletion. Thus these data are consistent with the hypothesis that theactivity of NHE3 in the kidney is required for maintenance of thenormal set point for Na -fluid volume balance.: i2 E& p) G& f" I1 \+ x

The genetic background of the mice used in this study differed slightlyfrom that of our previous studies ( 8, 9, 12, 21 ), in whichthe mice were an equal mix of 129SVJ and Black Swiss strains. Thetransgenic mice were prepared on an ICR background and then backcrossedwith Nhe3  / mice for two to three generationsbefore breeding pairs were established to generate the animals used inthese experiments. Half of the mice used in the dietary NaCl-loadingexperiments were derived from pairs that had been backcrossed for threegenerations and would have had a genetic background of only ~12.5%ICR; the remaining mice were ~25% ICR. It is conceivable that theaddition of some ICR genetic background onto the already mixed 129SVJand Black Swiss background might have made the mice hardier, thereby contributing to their improved ability (via a reduction in both thedegree of volume depletion and the consequent hypovolemic renalfailure) to tolerate a low-salt diet. However, this would probablyrequire the presence of numerous modifier loci because the majority ofthe mice would have lacked any given ICR locus. It seems highlyunlikely that the differences in genetic background between the mice inthis study and in our previous studies could be a significant factor inthe ability of the tg Nhe3 / mice to toleratea high-salt diet, which clearly involves a substantial increase inNa absorption from the gut.

Previous studies showed that both single-nephron GFR ( 12 )and whole kidney GFR ( 4, 8 ) are reduced in Nhe3 / mice and that NHE3-deficient kidneyshave intact TGF mechanisms ( 12 ). Analysis of renalfunction in the salt-loaded tg Nhe3 / micerevealed that ERPF and GFR were also significantly reduced relative tothat for tg Nhe3  /  mice and that both wereessentially the same in tg Nhe3 / mice fedeither a 1 or a 5% NaCl diet. If reduced perfusion pressure resultingfrom the hypovolemic state were a major factor in the reduced GFR, thendietary salt loading would have been expected to increase GFR. Althoughit is clear from the results of a previous study ( 8 ) thatsevere hypovolemia in nontransgenic Nhe3 / mice during dietary Na restriction leads to a furtherreduction in GFR and hypovolemic renal failure, the present resultssupport the view that the reduced GFR in NHE3-deficient mice occurs asa direct compensation for the absorptive defect in the proximal tubuleand is due to renal mechanisms such as TGF ( 12 ) and areduction in renal plasma flow.

Although the intestinal function of NHE2 was not a subject of thisinvestigation, it is interesting that a low level of NHE3 in the smallintestine was sufficient to absorb large quantities of NaCl when themice were salt loaded, whereas wild-type levels of NHE2 presentthroughout the intestinal tract provide little, if any, capacity forsalt loading. NHE2-deficient mice do not have diarrhea and exhibit noalterations in aldosterone levels or blood pressure, suggesting thatits absence does not impair intestinal or renal Na absorption ( 8, 9, 20 ). Studies of the intestinal phenotype of NHE3 and NHE2/NHE3 double-knockout mice revealed no evidence thatNHE2 compensates for the loss of NHE3 ( 5, 9 ). Using theNHE2-deficient mouse, other investigators also have been unable toidentify an absorptive function for NHE2 ( 13, 17 ). Inparotid glands, where NHE2 is expressed on apical membranes, targeted ablation of NHE2 impaired secretion ( 17 ), a resultopposite to what would be expected if NHE2 served an absorptivefunction. The results of the present study further support the viewthat NHE3 is the critical absorptive Na /H exchanger in the intestine and that NHE2 has little, if any, role inNa absorption.
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In summary, we used a transgenic approach to partially rescue theintestinal absorptive defect of Nhe3 / mice.When subjected to dietary Na restriction,tg Nhe3 / mice were able to reduce urinaryNa excretion to very low levels, consistent with the viewthat normalization of fluid delivery to the distal convoluted tubulevia a reduction in GFR ( 4, 12 ) largely prevents theoverloading of more distal mechanisms for Na reabsorptionand consequent salt wasting. After dietary salt loading, to partiallyalleviate the extracellular fluid volume deficit, GFR remained lower intg Nhe3 / mice than in wild-type controls,suggesting that reduced perfusion pressure resulting from systemichypovolemia is probably not a major factor in the reduced GFR.Therefore, the reduction is more likely the result of intrarenalhomeostatic mechanisms involving TGF and reduced renal plasma flow.Finally, after dietary salt loading that far exceeded the levelsoccurring in wild-type controls on a normal diet,tg Nhe3 / mice were still in a chronicvolume-depleted state, indicating that NHE3 in the kidney affects theset point for Na -fluid volume homeostasis.) z' M' E! W! }" v# _$ `$ j) x9 ~
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ACKNOWLEDGEMENTS6 I7 R3 y0 e* X) _5 J
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We thank Maureen Luehrmann and Angel Whitaker for expert animal husbandry.
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