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

Alison L. Woo1, William T. Noonan2, Patrick J. Schultheis3, Jonathan C. Neumann1, Patrice A. Manning1, John N. Lorenz2, and Gary E. Shull1

Departments of 1 Molecular Genetics, Biochemistry, and Microbiology and 2 Molecular and Cellular Physiology, The University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524; and 3 Department of Biological Sciences, Northern Kentucky University, Highland Heights, Kentucky 41099


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

sodium/hydrogen exchanger; diarrhea; Slc9a3; sodium absorption; sodium-fluid volume homeostasis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NA+/H+ exchanger isoform 3 (NHE3) is one of several Na+ transport proteins in renal epithelial cells that are involved in maintaining Na+-fluid volume homeostasis. Localized to apical membranes of the proximal tubule and to a lesser extent in the thick ascending limb of Henle, NHE3 transports Na+ into the cell in exchange for H+ and is responsible for absorbing large quantities of NaCl and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, with accompanying water (1, 3, 24). NHE3 null mutant (Nhe3-/-) mice have severe absorptive defects in both the kidney and intestine, and they exhibit characteristics of chronic volume depletion, including low blood pressure, high levels of renin mRNA in kidney, and high serum aldosterone (21). In situ microperfusion and micropuncture studies showed that reabsorption of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and water was reduced in the proximal tubule of Nhe3-/- mice (12, 24). However, fluid delivery to the distal convoluted tubule was not significantly different from that in wild-type mice, and this appeared to 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 in GFR might be a compensatory mechanism by which the kidneys of Nhe3-/- mice conserve Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (4, 8, 12).

The observed normalization of fluid delivery to the distal convoluted tubule of NHE3-deficient mice (12) raised the possibility that the proximal tubule absorptive defect itself might not lead to significant renal salt wasting. In a subsequent study, when subjected to dietary Na+ restriction, Nhe3-/- mice lost weight rapidly and did in fact exhibit urinary salt wasting (8), although apparently not as severe as in mice lacking transporters along some of the more distal segments of the nephron, such as the ROMK K+ channel (10) and Na+-K+-2Cl- cotransporter (23) of the thick ascending limb and the epithelial Na+ channel (ENaC) of the connecting tubule and collecting duct (2, 6, 14, 18, 23). By the third day of dietary Na+ restriction, however, many of the Nhe3-/- mice were undergoing hypovolemic renal failure. This raises the possibility that systemic volume depletion, and not just the loss of NHE3 in the kidney, might cause some degree of renal dysfunction. Thus extracellular fluid volume depletion itself, which is exacerbated by the diarrheal state during dietary Na+ restriction, may have contributed to the mild impairment in the ability of the NHE3-deficient kidney to retain Na+. Similarly, it was unclear whether the observed reduction in GFR in Nhe3-/- mice might have been due, in part, to systemic hypovolemia and hypotension rather than to an appropriate regulation of fluid delivery to the distal tubule via TGF mechanisms. Attempts to improve the fluid volume status of Nhe3-/- mice by feeding them a high-NaCl diet resulted in swelling of the intestine, severe hypovolemia, and death, further suggesting that the intestinal defect impaired extracellular fluid-volume homeostasis.

Thus the coexisting intestinal absorptive defect and chronic diarrhea in Nhe3-/- mice represent a major confounding factor in determining the specific effects of the loss of NHE3 in the kidney on renal Na+ conservation, GFR, and extracellular fluid-volume homeostasis. To assess these issues, we developed a transgenic mouse in which NHE3 is expressed in the small intestine via the intestinal fatty acid binding protein (IFABP) promoter and crossed it with Nhe3-/- mice. Transgenic Nhe3-/- (tgNhe3-/-) mice were then subjected to dietary Na+ restriction and Na+ loading, and renal function was analyzed. Both dietary Na+ restriction and Na+ loading were better tolerated in tgNhe3-/- mice than in nontransgenic Nhe3-/- mice. Salt loading led to a substantial reduction of aldosterone levels in tgNhe3-/- mice, indicating a partial correction of the extracellular fluid-volume deficit. However, tgNhe3-/- mice remained mildly hypotensive and had reduced GFR compared with Nhe3+/+ mice also harboring the IFABP-Nhe3 transgene (tgNhe3+/+).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Production and genotyping of mutant and transgenic mice. The rat NHE3 cDNA was cloned into an expression plasmid containing the small intestine-specific IFABP promoter (nucleotides -1178 to +28) and a t-intron polyadenylation cassette [provided by J. A. Whitsett (27)] (Fig. 1A). The IFABP/NHE3 construct was microinjected into fertilized oocytes from Institute of Cancer Research (ICR) mice, and injected oocytes were implanted into the uterus of pseudopregnant mice to produce transgenic animals by the University of Cincinnati transgenic core facility. Mice carrying the transgene integrated into their genome were identified by PCR analysis. The 5'-oligonucleotide primer sequence was from the IFABP promoter sequence (5'-CTGCCAGGTTATCTCTTGAAC-3'), and the 3' reverse primer 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, and 72°C for 30 s, followed by 72°C for 10 min. ICR transgenic mice were backcrossed for two to three generations with Nhe3+/- mice of a mixed 129SvJ and Black Swiss background (21) to produce Nhe3+/+ and Nhe3-/- mice carrying the IFABP/NHE3 transgene (tgNhe3+/+ and tgNhe3-/-). Thus the genetic background of the mice used in these experiments was 12.5-25% ICR, with the remainder being an equal mix of 129SVJ and Black Swiss. Nhe3+/- mice were generated and maintained as previously described (21). PCR genotyping was performed using the following primers: a forward primer corresponding to a sequence in exon 6 (5'-CTTTTGCGGCATCTGCTGTCAG-3'), a reverse primer corresponding to a sequence in intron 6 (5'-ACTACTAAGAGTGCTCCTAGCTCTCACC-3'), and a reverse primer corresponding 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, and 72°C for 30 s. The experiments described below were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee at the University of Cincinnati College of Medicine. All experimental pairs of tgNhe3-/- and tgNhe3+/+ mice were 8-12 wk old and were littermates matched by both age and sex to ensure that no strain, age, or sex biases contributed to physiological outputs.


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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 acid binding protein (IFABP) promoter to drive expression of rat NHE3 cDNA in small intestine. Arrows represent PCR primers used to identify mice with genomic insertion of the transgene. B: PCR genotyping of transgenic mice showing presence or absence of a 310-bp product in transgenic (+) or nontransgenic (-) mice, respectively. H2O 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 endogenous NHE3 mRNA is 5.6 kb, the transgene mRNA is 3.5 kb. D: Western blot analysis of NHE3 in small intestine of transgenic and nontransgenic Nhe3+/+ and Nhe3-/- mice, using 20 µg of total membranes and a rat anti-NHE3 antibody.

RNA isolation and Northern blot analysis. Total RNA was extracted from tissues using Tri-Reagent (Molecular Research Center). Total RNA (10 µg/sample) was mixed with Glyoxal Sample Buffer (BioWhittaker Molecular Applications, Rockland, ME), separated by electrophoresis in 1% agarose, and transferred to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ). Northern blots were screened using [32P]-labeled cDNA probes 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 L32 ribosomal protein mRNA as a loading control and reported as mean volume integrated values for four pairs of tgNhe3-/- and tgNhe3+/+ mice on 1 or 5% NaCl diets.

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 protease inhibitor 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 the pellet was homogenized and centrifuged again. The combined supernatants were centrifuged at 200,000 g for 1 h at 4°C, and the pellet containing total membranes was resuspended in homogenization buffer. A BCA protein assay kit (Pierce, Rockford, IL) was used to quantitate protein concentrations. Total membrane preparations were analyzed by Western blot analyses using 1 µg/ml of the rabbit NHE3 polyclonal antibody (Chemicon, Temecula, CA), as described previously (26). Signal detection was accomplished using the SuperSignal 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. The contents of each segment were removed, and the weights of the tissue and contents were recorded. Contents were mixed with 1 ml sterile saline and centrifuged, and the pH of the supernatant was recorded.

Balance studies using varying Na+-content diets. Mice were housed in metabolic cages and provided with drinking water and food ad libitum, as described previously (22). Food (Harlan Teklad, Madison, WI) contained normal (1%), low (0.01%), or high (5%) levels of NaCl. Body weights and the amount of food and water consumed were recorded every day. Urine samples were collected daily and were processed and analyzed for Na+ and K+ concentrations as described previously (8).

Serum aldosterone levels. Mice were anesthetized, and blood was drawn by cardiac puncture. Serum was separated from blood cells and stored at -20°C. Serum samples were diluted 1:4 in sterile PBS, and aldosterone concentrations were determined using an RIA kit (Diagnostic Products, Los Angeles, CA).

Renal hemodynamic measurements. Baseline renal function was determined in two groups of seven pairs of tgNhe3+/+ and tgNhe3-/- mice maintained on either a 1 or a 5% NaCl diet for 5 days. Mice were anesthetized with ketamine (50 µg/g body wt) and inactin (100 µg/g body wt) and surgically instrumented for renal measurements as described previously (12). Immediately after surgery, a bolus (3 µl/g body wt) of 1% FITC-inulin and 3% PAH in isotonic saline was administered. This was followed by a maintenance infusion of the same solution at 0.15 µl · min-1 · g body wt-1. After a 30-min equilibration period, baseline renal function was determined through two 30-min urine samples collected through a catheter in the bladder (12). At the midpoint of each baseline collection, an arterial blood sample (60 µl) was obtained for determination of plasma FITC-inulin (11) and PAH (25) concentrations, and donor blood was administered to replace the lost volume after each sample was obtained. At the end of the second baseline collection, another blood sample was acquired and plasma electrolyte levels were measured using a pH/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, and effective renal plasma flow (ERPF) was calculated from PAH clearance.

Statistics. Statistical analysis was performed by either Student's t-test or analysis of variance. When analysis of variance was applied, either a single-factor design or a mixed-factorial design with repeated measures on the second factor was used, and individual contrasts were used to compare individual group means when needed. Data are presented as means ± SE, and statistical significance was regarded as P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of transgenic mice expressing NHE3 in the small intestine. No promoters are available that would allow transgenic expression of NHE3 throughout the intestinal tract. To partially rescue the intestinal absorptive defect of Nhe3-/- mice, we used the rat IFABP promoter to drive expression of a rat NHE3 transgene (Fig. 1A) in the small intestine. Genomic integration of the transgene was detected by PCR analysis (Fig. 1B). Northern blot analysis of kidney, small intestine, cecum, and colon tissue from tgNhe3+/+ mice revealed expression of a 3.5-kb mRNA corresponding to the transgene only in small intestine, whereas the 5.6-kb mouse NHE3 mRNA was expressed in all tissues (Fig. 1C). The transgenic mice were then bred with Nhe3+/- mice. Western blot analysis demonstrated that NHE3 protein was expressed in kidneys (data not shown) and small intestines (Fig. 1D) of both transgenic and nontransgenic wild-type mice but not in the kidneys (data not shown) of tgNhe3-/- mice. In tgNhe3-/- mice, NHE3 protein was expressed in the small intestine, although at a lower level than in wild-type controls (Fig. 1D). In addition to a protein corresponding to full-length rat NHE3, a smaller band of unknown identity was also detected in tgNhe3-/- small intestine.

Analysis of intestinal weight and intestinal contents. The loss of NHE3 in the intestinal tract causes a severe absorptive defect, resulting in chronic diarrhea. All segments of the intestine are enlarged in Nhe3-/- mice, and the volume and pH of the luminal contents are increased (21). Expression of functional NHE3 in the Nhe3-/- small intestine (Fig. 1, C and D) would be expected to at least partially alleviate these defects in this segment. Gross examination of the tgNhe3-/- intestinal tract revealed a less bloated small intestine, whereas the cecum and colon appeared similar to those of Nhe3-/- mice (21). The weight of the small intestine was greater in tgNhe3-/- mice than in tgNhe3+/+ mice; however, the weight and pH of the small intestinal contents in tgNhe3-/- mice were not significantly different from those in tgNhe3+/+ mice (Fig. 2). On the other hand, the weights and luminal contents of the tgNhe3-/- cecum and colon, where the transgene was not expressed, were significantly greater than in tgNhe3+/+ mice, and the pH of the luminal contents was significantly more alkaline (Fig. 2). These data suggest that the observed levels of expression of the NHE3 transgene partially restored the absorptive capabilities of the small intestine.


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Fig. 2.   Analysis of intestinal segments and contents of transgenic (tg)Nhe3+/+ and tgNhe3-/- mice. A: weight of small intestine, 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, and colon. Values are means ± SE; n = 6/genotype. *P < 0.05 compared with corresponding tgNhe3+/+ values.

tgNhe3-/- mice exhibit increased tolerance for a Na+-restricted diet. When Nhe3-/- mice not carrying the transgene were fed a Na+-restricted (0.01% NaCl) diet, they exhibited severe weight loss and renal salt wasting (8). To test the Na+-handling capabilities of tgNhe3-/- mice, tgNhe3+/+ and tgNhe3-/- 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), tgNhe3-/- mice had lower urinary Na+ excretion compared with tgNhe3+/+ mice, consistent with only a partial rescue of the intestinal phenotype. However, during dietary Na+ restriction, tgNhe3-/- mice lost only 6% of their body weight (Table 1), which was a major improvement over the 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 excrete substantial amounts of Na+ even after 3 days on low salt (8), tgNhe3-/- mice lowered their Na+ excretion after 3 days to very low levels that were not significantly different from that for tgNhe3+/+ mice (Fig. 3A, Table 1).

                              
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Table 1.   Balance studies in tgNhe3+/+ and tgNhe3-/- mice on a 0.01% NaCl diet



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Fig. 3.   Urinary Na+ excretion by tgNhe3+/+ and tgNhe3-/- 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 < 0.001 compared with corresponding tgNhe3+/+ values.

Some of the metabolic characteristics of tgNhe3-/- mice during dietary Na+ restriction were similar to those reported for nonrescued Nhe3-/- mice (8). Compared with tgNhe3+/+ controls, tgNhe3-/- mice drank significantly more water regardless of diet, and they had a greater urinary output when fed a 0.01% NaCl diet (Table 1). Urinary K+ excretion was lower in tgNhe3-/- mice, although the difference was not statistically significant and was likely related to increased intestinal K+ secretion as a result of the diarrheal state. Consistent recovery of fecal samples from tgNhe3-/- mice was not possible due to the persistent diarrhea resulting from the absorptive defect in the cecum and colon.

tgNhe3-/- mice fed a 5% NaCl diet have increased urinary Na+ excretion indicative of increased intestinal NaCl absorption. NaCl loading via continuous infusion of saline through a venous catheter can restore extracellular fluid volume in Nhe3-/- mice, as shown by increases in blood pressure to a level similar to that for Nhe3+/+ mice (Lorenz JN, unpublished observations). This suggested that it might be possible to restore extracellular fluid volume by dietary NaCl loading. However, when Nhe3-/- mice not carrying the transgene were fed a high-salt diet (5% NaCl), their small intestines became severely swollen, probably due to an osmotic effect from the high levels of NaCl in the gut, and they died within 48 h (Lorenz JN and Shull GE, unpublished observations). In contrast, when tgNhe3-/- mice were fed a normal-salt diet (1% NaCl) for 3 days followed by a 5% NaCl diet for 4 days, they not only tolerated the high-salt diet, but their urinary Na+ excretion increased to levels higher than that seen in tgNhe3+/+ mice maintained on a normal diet (Fig. 3B, Table 2). These data demonstrate, importantly, that the intestinally rescued NHE3-deficient mice can compensate for possible urinary NaCl losses through increases in intestinal NaCl absorption, whereas their nonrescued counterparts could not. There was no difference in weight between tgNhe3-/- and tgNhe3+/+ mice fed either diet, although body weight decreased slightly in both genotypes when on the 5% NaCl diet (Table 2). Again, tgNhe3-/- mice drank more water than tgNhe3+/+ mice regardless of diet, and both genotypes consumed more water and increased their urinary output 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 increased intestinal Na+ absorption in tgNhe3-/- mice.

                              
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Table 2.   Balance studies in tgNhe3+/+ and tgNhe3-/- mice on a 5% NaCl diet

Serum aldosterone and renin mRNA levels in the kidney are decreased in tgNhe3-/- mice fed a 5% NaCl diet. The major hormonal mechanism for correction of a deficit in extracellular fluid volume is an increase in serum aldosterone, which stimulates the absorption of NaCl in the kidney and intestine. When fed a diet containing 1% NaCl, tgNhe3-/- mice had a serum aldosterone level that was similar to that observed previously in Nhe3-/- mice (21) and was 11-fold greater than that in tgNhe3+/+ mice (Fig. 4). These data indicate that tgNhe3-/- mice fed a 1% NaCl diet have a severe deficit in extracellular fluid volume. Serum aldosterone was sharply decreased in tgNhe3-/- mice when they were fed a 5% NaCl diet, although it was still higher than that in tgNhe3+/+ mice (Fig. 4), indicating that the deficit in extracellular fluid volume can be partially corrected by dietary salt loading.


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Fig. 4.   Serum aldosterone levels in tgNhe3+/+ and tgNhe3-/- 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 < 0.05 compared with corresponding tgNhe3+/+ values. dagger P < 0.05 compared with 1% NaCl diet.

Volume depletion also activates the intrarenal renin-angiotensin system, as indicated by an increase in the expression of renin mRNA in the kidney in response to dietary Na+ restriction (7). As shown in Fig. 5, renin mRNA levels were about sevenfold greater in tgNhe3-/- kidneys than in tgNhe3+/+ 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 the volume status of tgNhe3-/- mice was improved by dietary salt loading.


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Fig. 5.   Northern blot analysis of renin mRNA in kidneys from tgNhe3+/+ and tgNhe3-/- mice. A: blots containing RNA samples (10 µg/lane) from individual kidneys from 4 pairs of mice on a 1% NaCl diet and 4 pairs of mice on a 5% NaCl diet were hybridized with a renin probe and then stripped and hybridized with a probe for the ribosomal L32 protein (loading control). B: renin mRNA hybridization signals were quantitated by PhosphorImager analysis and normalized to the signal for L32 mRNA in that sample. Fold-changes in tgNhe3-/- samples relative to tgNhe3+/+ samples are represented as means ± SE. *P < 0.02 compared with 1% NaCl diet.

Renal function in tgNhe3-/- mice fed a 1 or 5% NaCl diet. After 5 days on either a 1 or a 5% NaCl diet, tgNhe3+/+ and tgNhe3-/- mice were anesthetized and surgically prepared for analysis of renal function, blood pressure and heart rate, and collection of blood samples. Under anesthesia, mean arterial pressure (Fig. 6A) was lower in tgNhe3-/- mice than in tgNhe3+/+ mice regardless of diet, but administration of the high-salt diet increased blood pressure in tgNhe3-/- mice (88.4 ± 3.2 compared with 77.0 ± 4.6 mmHg when fed the 1% NaCl diet), whereas it had no effect in tgNhe3+/+ mice. There were no significant differences in heart rate between any of the groups (Fig. 6B). Also, in tgNhe3+/+ mice, the hematocrit did not change when animals were placed on the high-salt diet, but in tgNhe3-/- animals, the 5% NaCl diet significantly reduced the hematocrit (Table 3). The effects of high-salt intake on blood pressure and hematocrit are consistent with a partial correction of the extracellular fluid volume deficit in NHE3-deficient mice expressing the NHE3 transgene in the small intestine. None of the groups differed with respect to plasma Na+ or arterial blood HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, but blood pH was significantly lower in tgNhe3-/- mice regardless of diet (Table 3). Plasma K+ increased significantly in tgNhe3+/+ mice on the 5% NaCl diet compared with the same genotype on the 1% NaCl diet, but this increase did not occur in tgNhe3-/- mice (Table 3).


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Fig. 6.   Mean arterial pressure (A) and heart rate (B) of tgNhe3+/+ and tgNhe3-/- mice fed either a 1 or a 5% NaCl diet. Groups of 7 experimental pairs were fed each of the diets for 5 days, anesthetized, and surgically instrumented for analysis of cardiovascular parameters (shown here), blood, and renal function (shown in Table 3). *P < 0.01 compared with corresponding tgNhe3+/+ values.


                              
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Table 3.   Blood and renal measurements in tgNhe3+/+ and tgNhe3-/- mice on a 1 or 5% NaCl diet

GFR and ERPF were lower in tgNhe3-/- mice than in tgNhe3+/+ mice, and the effect was independent of diet (Table 3). Furthermore, administration of a high-salt diet did not alter GFR in either group. Renal plasma flow, on the other hand, increased in response to the high-salt diet in tgNhe3+/+ animals but not in tgNhe3-/- animals. Urinary flow rate was not different between the groups and increased comparably in both groups of mice in response to high-NaCl intake. The amounts of filtered Na+ and K+ were significantly less in tgNhe3-/- mice on either diet, consistent with the reduction in GFR, but the filtered load did not change in response to a high-NaCl diet in either group. Na+ excretion and fractional Na+ excretion were both lower in tgNhe3-/- mice than in tgNhe3+/+ mice, regardless of diet (Table 3), and the sodium excretory response to a high-salt diet was the same in both genotypes. There were no significant differences in K+ excretion among any of the groups, but fractional K+ excretion was significantly reduced in tgNhe3+/+ mice on the 5% NaCl diet compared with tgNhe3+/+ mice on the 1% NaCl diet, consistent with increased plasma K+.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies demonstrated that both Na+ reabsorption in the proximal tubule and systemic Na+-fluid volume homeostasis are severely perturbed in Nhe3-/- mice (8, 12, 21, 24) and that partial compensation for the renal absorptive defect occurs by a reduction in GFR (4, 8, 12). However, Nhe3-/- mice also have an intestinal absorptive defect, which clearly contributes to volume depletion during dietary Na+ restriction and might also play a role in volume depletion under normal conditions. This makes it difficult to accurately assess 1) the capacity of the NHE3-deficient kidney to recover NaCl; 2) the degree to which the reduction in GFR represents direct compensation for the proximal tubule absorptive 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 chronic extracellular fluid volume depletion.

To resolve these issues, we generated Nhe3-/- mice expressing NHE3 in the small intestine with the expectation that this would allow dietary salt loading, an approach that was not successful with nontransgenic Nhe3-/- mice, apparently because of the osmotic effects of high salt in the lumen of the gut. It would have been preferable to express NHE3 in small intestine, cecum, and colon; however, no promoters were available that would allow this. Transgenic expression of rat NHE3 under the control of the IFABP promoter yielded lower levels of expression than that of the endogenous protein, and expression was limited to the small intestine and therefore did not correct the absorptive defect in the cecum or colon. In addition to an NHE3 protein corresponding in size to wild-type NHE3, there was a diffuse product of lower molecular weight in the tgNhe3-/- small intestine; the identity of this smaller product is unclear. It is possible that glycosylation or trafficking of NHE3, expressed from the transgene, is inefficient in the Golgi complex of mouse small intestinal epithelial cells. An alternative possibility is that the rat NHE3 mRNA derived from the transgene, which lacks most of the wild-type 5'- and 3'-untranslated sequences, may not be efficiently localized to endoplasmic reticulum-bound ribosomes, as there is evidence that untranslated mRNA sequences might be important in this process (16). Either of these possibilities might explain the apparently smaller or partially degraded NHE3 and the discrepancy between the amount of mRNA expressed from the transgene and the relatively low amount of normal NHE3 protein detected. Nevertheless, these low levels of NHE3 expression did lead to normalization of the pH of small intestinal contents and, as discussed below, allowed a substantial amount of salt loading when the mice were fed a 5% NaCl diet, thereby eliminating the diarrheal state as a major factor in extracellular fluid volume depletion.

When the mice were maintained on a 1% NaCl diet, transgenic expression of NHE3 in the small intestine of Nhe3-/- mice did not appear to improve extracellular fluid volume status, as they had elevated levels of serum aldosterone, highly induced renin mRNA in kidney, and low blood pressure similar to that seen in nontransgenic Nhe3-/- mice (21). The net intestinal absorption and urinary excretion of NaCl (which must be equivalent when the mice are in balance) corresponded to ~0.22 and ~2.1 ml of isotonic fluid/day for tgNhe3-/- and tgNhe3+/+ mice, respectively, consistent with the possibility that poor absorption of NaCl from the intestinal tract was a major factor in systemic volume depletion. Surprisingly, in response to dietary Na+ restriction, tgNhe3-/- mice exhibited little evidence of urinary salt wasting. The relatively small reduction in body weight, relative to that observed earlier for Na+-restricted Nhe3-/- mice (8), is probably due primarily to intestinal losses of NaCl because urinary losses on days 2 and 3 of Na+ restriction were only equivalent to ~0.015 ml of isotonic fluid/day. These results indicate that the NHE3-deficient kidney has a substantial ability to recover NaCl and suggest that the hypovolemic renal failure observed in our previous study after 3 days of dietary Na+ restriction (8), which was a likely factor in the observed renal salt wasting, was brought on largely by the continuing intestinal losses of salt and water.

When the mice were maintained on a 5% NaCl diet, urinary Na+ excretion in tgNhe3-/- mice increased to a level ~18 times that in tgNhe3-/- mice on a normal 1% NaCl diet and ~2.5 times that in tgNhe3+/+ mice on a 1% NaCl diet. The net urinary excretion (and intestinal absorption) of NaCl per day in tgNhe3-/- mice was equivalent to that in ~4.3 ml of extracellular fluid and was far in excess of that excreted by the NHE3-deficient kidney when the mice were fed either a normal or a Na+-restricted diet (Tables 1 and 2). This indicates that the effects of the intestinal absorptive defect on extracellular fluid volume homeostasis can be overcome by dietary salt loading in these animals. As shown by the sharply reduced serum aldosterone levels, the decrease in the level of induction of renin mRNA in the kidney, and the increase in mean arterial pressure, the extracellular fluid volume status of tgNhe3-/- mice was substantially improved when they were fed the 5% NaCl diet. Nevertheless, serum aldosterone and kidney renin mRNA were still elevated and blood pressure was still reduced relative to that for tgNhe3+/+ mice. These data suggest that the absence of NHE3 in the kidney, even when the mice are subjected to dietary NaCl loading, results in a certain degree of chronic volume depletion. Thus these data are consistent with the hypothesis that the activity of NHE3 in the kidney is required for maintenance of the normal set point for Na+-fluid volume balance.

The genetic background of the mice used in this study differed slightly from that of our previous studies (8, 9, 12, 21), in which the mice were an equal mix of 129SVJ and Black Swiss strains. The transgenic mice were prepared on an ICR background and then backcrossed with Nhe3+/- mice for two to three generations before breeding pairs were established to generate the animals used in these experiments. Half of the mice used in the dietary NaCl-loading experiments were derived from pairs that had been backcrossed for three generations and would have had a genetic background of only ~12.5% ICR; the remaining mice were ~25% ICR. It is conceivable that the addition of some ICR genetic background onto the already mixed 129SVJ and Black Swiss background might have made the mice hardier, thereby contributing to their improved ability (via a reduction in both the degree of volume depletion and the consequent hypovolemic renal failure) to tolerate a low-salt diet. However, this would probably require the presence of numerous modifier loci because the majority of the mice would have lacked any given ICR locus. It seems highly unlikely that the differences in genetic background between the mice in this study and in our previous studies could be a significant factor in the ability of the tgNhe3-/- mice to tolerate a high-salt diet, which clearly involves a substantial increase in Na+ 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 kidneys have intact TGF mechanisms (12). Analysis of renal function in the salt-loaded tgNhe3-/- mice revealed that ERPF and GFR were also significantly reduced relative to that for tgNhe3+/+ mice and that both were essentially the same in tgNhe3-/- mice fed either a 1 or a 5% NaCl diet. If reduced perfusion pressure resulting from the hypovolemic state were a major factor in the reduced GFR, then dietary salt loading would have been expected to increase GFR. Although it is clear from the results of a previous study (8) that severe hypovolemia in nontransgenic Nhe3-/- mice during dietary Na+ restriction leads to a further reduction in GFR and hypovolemic renal failure, the present results support the view that the reduced GFR in NHE3-deficient mice occurs as a direct compensation for the absorptive defect in the proximal tubule and is due to renal mechanisms such as TGF (12) and a reduction in renal plasma flow.

Although the intestinal function of NHE2 was not a subject of this investigation, it is interesting that a low level of NHE3 in the small intestine was sufficient to absorb large quantities of NaCl when the mice were salt loaded, whereas wild-type levels of NHE2 present throughout the intestinal tract provide little, if any, capacity for salt loading. NHE2-deficient mice do not have diarrhea and exhibit no alterations in aldosterone levels or blood pressure, suggesting that its 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 that NHE2 compensates for the loss of NHE3 (5, 9). Using the NHE2-deficient mouse, other investigators also have been unable to identify an absorptive function for NHE2 (13, 17). In parotid glands, where NHE2 is expressed on apical membranes, targeted ablation of NHE2 impaired secretion (17), a result opposite to what would be expected if NHE2 served an absorptive function. The results of the present study further support the view that NHE3 is the critical absorptive Na+/H+ exchanger in the intestine and that NHE2 has little, if any, role in Na+ absorption.

In summary, we used a transgenic approach to partially rescue the intestinal absorptive defect of Nhe3-/- mice. When subjected to dietary Na+ restriction, tgNhe3-/- mice were able to reduce urinary Na+ excretion to very low levels, consistent with the view that normalization of fluid delivery to the distal convoluted tubule via a reduction in GFR (4, 12) largely prevents the overloading of more distal mechanisms for Na+ reabsorption and consequent salt wasting. After dietary salt loading, to partially alleviate the extracellular fluid volume deficit, GFR remained lower in tgNhe3-/- mice than in wild-type controls, suggesting that reduced perfusion pressure resulting from systemic hypovolemia is probably not a major factor in the reduced GFR. Therefore, the reduction is more likely the result of intrarenal homeostatic mechanisms involving TGF and reduced renal plasma flow. Finally, after dietary salt loading that far exceeded the levels occurring in wild-type controls on a normal diet, tgNhe3-/- mice were still in a chronic volume-depleted state, indicating that NHE3 in the kidney affects the set point for Na+-fluid volume homeostasis.


    ACKNOWLEDGEMENTS

We thank Maureen Luehrmann and Angel Whitaker for expert animal husbandry.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-50594, DK-57552, HL-61974, and T32-DK-07727.

Address for reprint requests and other correspondence: G. E. Shull; Dept. of Molecular Genetics, Biochemistry, and Microbiology; Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0524 (E-mail: shullge{at}ucmail.uc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 11, 2003;10.1152/ajprenal.00418.2002

Received 3 December 2002; accepted in final form 5 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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