Proximal tubule Na+/H+ exchanger activity in adult NHE8−/−, NHE3−/−, and NHE3−/−/NHE8−/− mice

Michel Baum, Katherine Twombley, Jyothsna Gattineni, Catherine Joseph, Lin Wang, Qiuyu Zhang, Vangipuram Dwarakanath, Orson W. Moe

Abstract

NHE3 is the predominant Na+/H+ exchanger on the brush-border membrane (BBM) of the proximal tubule in adults. However, NHE3 null mice still have significant renal BBM Na+/H+ activity. NHE8 has been localized to the BBM of proximal tubules and is more highly expressed in neonates than adult animals. The relative role of NHE8 in adult renal H+ transport is unclear. This study examined whether there was compensation by NHE8 in NHE3−/− mice and by NHE3 in NHE8−/− mice. NHE3−/− mice had significant metabolic acidosis, and renal BBM NHE8 protein abundance was greater in NHE3−/− mice than control mice, indicating that there may be compensation by NHE8 in NHE3−/− mice. NHE8−/− mice had serum bicarbonate levels and pH that were not different from controls. NHE3 protein expression on the BBM was greater in NHE8−/− mice than in wild-type mice, indicating that there may be compensation by NHE3 in NHE8−/− mice. Both BBM NHE3 and NHE8 protein abundance increased in response to acidosis. Blood pressure and Na+/H+ exchanger activity were comparable in NHE8−/− mice to that of controls, but both were significantly lower in NHE3−/− mice compared with control mice. Compared with NHE3−/− mice, NHE3−/−/NHE8−/− mice had lower blood pressures. While serum bicarbonate was comparable in NHE3−/− mice and NHE3−/−/NHE8−/− mice, proximal tubule Na+/H+ exchange activity was less in NHE3−/−/NHE8−/− mice compared with NHE3−/− mice. In conclusion, NHE3 is the predominant Na+/H+ exchanger in adult mice. NHE8 may play a compensatory role in renal acidification and blood pressure regulation in NHE3−/− mice.

  • sodium/hydrogen exchanger
  • proximal tubule
  • development
  • acidosis

the proximal tubule reabsorbs most of the filtered bicarbonate, which is predominantly mediated by apical membrane Na+/H+ exchange (5, 21, 31, 33). The Na+/H+ exchanger also plays an important role in mediating proximal tubule NaCl transport (19, 22, 23, 31, 32). From an ultrafiltrate of plasma, the proximal tubule apical membrane Na+/H+ exchanger secretes H+ for bicarbonate reclamation and acid excretion and also controls base absorption indirectly by titrating the valence of citrate. The bicarbonate absorption generates a high chloride-low bicarbonate solution from which chloride is reabsorbed both actively via the parallel action of Na+/H+ and chloride/base exchangers and passively via paracellular diffusion (3, 22). In the adult rodent, the predominant apical membrane Na+/H+ exchanger is NHE3 (7, 8, 23, 31, 33).

The importance of NHE3 in proximal tubule volume and NaCl transport has been facilitated by studying NHE3−/− mice (11, 18, 19, 23, 31, 32). NHE3−/− mice have a 50% reduction in the rate of volume absorption from the glomerular ultrafiltrate (19). The rate of NaCl transport from a high chloride-low bicarbonate solution simulating late proximal tubular fluid is lower in NHE3−/− mice, and the formate-stimulated component of transcellular chloride/base exchange is virtually abolished in NHE3−/− mice (32).

Adult NHE3−/− mice have a mild metabolic acidosis with a serum bicarbonate only 3–5 meq/l less than control mice (23, 31). The rate of proximal tubule bicarbonate absorption in NHE3−/− mice is ∼30–40% that of control mice (23, 31). Approximately half of the residual bicarbonate absorption in NHE3−/− mice is due to the apical membrane H+-ATPase (31). In addition, there is evidence for the contribution of another amiloride-sensitive Na+/H+ exchanger in the proximal tubule that contributes significantly to Na+/H+ exchange activity in NHE3−/− mice (12).

NHE8 was cloned by Goyal et al. (15, 16) and localized to the brush border membrane (BBM) of the proximal tubule (15, 16). We have previously shown that NHE8 is relatively highly expressed on the proximal tubule BBM of neonatal mice and rats at a time when there is relatively low expression of NHE3 (7, 28). The apical membrane expression of NHE3 increases during postnatal development, reaching adult levels at the time of weaning concomitant with the decrease in apical expression of NHE8 (7, 28).

Presently, it is unclear whether NHE8 plays any role in adult proximal tubule renal acidification. In addition, it is unclear whether NHE8 and NHE3 compensate when the other isoform is deleted. The purpose of these studies was to examine whether NHE8 is upregulated in NHE3−/− mice and whether NHE3 is upregulated in NHE8−/− mice. We also examined Na+/H+ exchanger activity, serum bicarbonate, and blood pressure in adult NHE8−/−, NHE3−/−, and the double-deleted NHE3−/−/NHE8−/− mice to determine the relative roles of these transporters in proximal tubule acidification and hemodynamics.

METHODS

Animals.

NHE8−/− mice were generated by deletion of base 631–656 of NHE8 and replaced with the LacZ-Neo gene (Deltagen, San Mateo, CA). NHE3−/− mice were a gift from Dr. Gary Shull and were previously used by our laboratory (12, 23). In experiments directly comparing NHE3 and NHE8 null mice, NHE3 and NHE8 heterozygotes were mated to generate NHE3+/+/NHE8+/+, NHE3+/+/NHE8−/−, NHE3−/−/NHE8+/+, and NHE3−/−/NHE8−/− mice, and littermates of these heterozygote matings were compared. The mice were genotyped using tail clippings and were studied at 4–6 wk of age. All protocols were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center and were in accordance with the American Physiological Society's “Guiding Principles for Research Involving Animals and Human Beings.”

Phlebotomy and blood analysis.

Blood samples were obtained from mice anesthetized with Inactin (1 mg/10 g body wt, Sigma, St. Louis, MO) or inhaled isoflurane. One hundred microliters of blood was collected via retroorbital bleed or cardiac puncture and placed in heparinized tubes for analysis of electrolytes, pH, Pco2, and bicarbonate levels using a Stat Profile Critical Care Express blood-gas machine (Nova Biomedical, Waltham, MA).

RNA isolation and RNA blotting.

Renal cortex from control and NHE8−/− mice was homogenized in RNAzol [1:1, phenol-RNAzol (4 M guanidine thiocyanate, 25 mM disodium-citrate, pH 7.0), 0.5% sarcosyl] containing 3.6 μl/ml β-mercaptoethanol as previously described (7). RNA was extracted with chloroform and 3 M sodium acetate (pH 4.0), purified using isopropanol precipitation, and subsequently washed with 80% ethanol. Poly(A)+ RNA was purified using oligo (dT) column chromatography. Poly(A)+ RNA (5 μg) was fractionated using agarose-formaldehyde gel electrophoresis and transferred to a nylon filter (GeneScreen Plus; New England Nuclear, Boston, MA). The filter was prehybridized at 42°C for 4 h with ULTRAhyb buffer (Ambion, Austin, TX) and then hybridized to double-stranded uniform 32P-labeled cDNA probes at 42°C. The probes were synthesized by the random hexamer method using 100 ng of cDNA. The probe for NHE8 was a 2.5-kb EcoRI fragment (16), and for the β-actin probe a 1.5-kb EcoR1 fragment. The filter was then washed, and mRNA was detected using autoradiography.

Total protein and BBM vesicle isolation.

The kidneys were removed and placed in an ice-cold isolation buffer containing 300 mmol/l mannitol, 16 mmol/l HEPES, 5 mmol/l EGTA titrated to pH 7.4 with Tris containing PMSF (100 μg/ml), and 1 μl/ml protease inhibitor cocktail (Sigma). Kidneys were homogenized at 4°C to generate the total protein homogenate. BBM vesicles (BBMV) were isolated from the homogenate by differential centrifugation and magnesium precipitation as previously described (7). The final BBMV fraction was resuspended in isolation buffer. Protein fractions were assayed using the Bradford reaction with BSA as the standard (10).

SDS-PAGE and immunoblotting.

Protein samples were diluted in 5× loading buffer [5 mM Tris·HCl (pH 6.8), 10% β-mercaptoethanol, 10% glycerol, and 1% SDS; 50 μg/lane] and heated for 5 min at 85°C for NHE3 and 37°C for NHE8. The proteins were separated on an 8% polyacrylamide gel using SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Immobilon, Millipore, Billerica, MA) at 400 mA at 4°C for 1 h as described by our laboratory (7, 9). The blots were incubated in Blotto (1% nonfat milk and 0.05% Tween 20 in PBS) for at least 1 h before incubation with a primary antibody to NHE3 (3H3) or NHE8 (7A11) (9, 15). The blots were incubated in Blotto with the primary antibody and gently shaken overnight at 4°C. The blots were washed in PBS containing 0.1% Tween followed by incubation at room temperature with the secondary antibody, horseradish peroxidase-conjugated donkey anti-mouse antibody at a 1:15,000 dilution in 1% Blotto. Enhanced chemiluminescence was used to detect bound antibody (Amersham Biosciences, Piscataway, NJ). Confirmation of equal loading of protein was verified using an antibody to β-actin at 1:15,000 dilution (Sigma). Relative NHE3 and NHE8 protein abundance relative to β-actin was quantitated using densitometry. All signals for densitometry were performed using film exposures that were in the linear range for the amount of protein loaded.

cDNA synthesis and real-time PCR.

RNA was isolated from mice using the GenElute Mammalian Total RNA Miniprep Kit per the manufacturer's instructions (Sigma). The quality of the RNA obtained was assayed using an Epoch microplate spectrophotometer at 260- and 280-nm wavelengths (Biotek, Winooski, VT). RNA (2 μg) was first treated with DNAse I (Invitrogen, Carlsbad, CA), and the product was used to synthesize cDNA using random hexamer primers and reverse transcriptase (Stratagene, La Jolla, CA) as previously described (28).

Real-time PCR was performed using an iCycler PCR Thermal Cycler (Bio-Rad, Hercules, CA) to quantify relative mRNA abundance. For mouse studies, the primers for NHE8 (forward 5′-ACA GTT TCG CAT TTG GCT CCC TG-3′ and reverse 5′-TGT TGG TGA GGA CGA TGG AGA CTG-3′) were mixed with cDNA and SYBR Green Master Mix per the manufacturer's instructions (Bio-Rad). For NHE3, the primers used were (forward 5′-TTC AAA TGG CAC CAC GTC CAG G-3′ and reverse 5′-TGA CTT TGT GGG ACA GTT GAA AG-3′). The PCR conditions were denaturation at 94°C for 30 s, annealing at 61°C for 20 s, and extension at 72°C for 20 s for 40 cycles. The housekeeping 28S rRNA (forward 5′-TTG AAA ATC CGG GGG AGA G-3′ and reverse 5′-ACA TTG TTC CAT GCC AG-3′) was used to normalize the relative expression of NHE8 using the method described by Vandesompele et al. (29).

In vitro microperfusion measurement of proximal convoluted Na+/H+ exchange activity.

Kidneys were removed from NHE8+/+/NHE3+/+, NHE3+/+/NHE8−/−, NHE3−/−, /NHE8+/+, and NHE3+/+/NHE8−/− mice and placed in ice-cold Hanks' solution. Hanks' solution contained (in mM) 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4, 1 MgCl2, 10 Tris (hydroxymethyl) amino methane hydrochloride, 0.25 CaCl2, 2 glutamine, and 2 l-lactate (pH 7.4). Proximal convoluted tubules were dissected free hand in Hanks' solution without collagenase (12).

Proximal convoluted tubules were perfused using concentric glass pipettes with a sodium-containing solution (solution B) and bathed with a solution containing (in mM) 115 NaCl, 25 NaHCO3, 10 Na acetate, 8.3 glucose, 5 KCl, 4.0 Na2HPO4, 1.8 mM CaCl2, 1 MgSO4, 5 alanine, 2 glutamine, and 2 lactate. The composition of the solutions used to assay Na+/H+ exchange are listed in Table 1. A Nikon Eclipse TE 2000-U inverted epifluorescent microscope equipped with a Photometrics Cascade:512F microscopy camera (Ottobrunn, Germany) and Lambda DG-4/DG-5 illumination system (Sutter Instrument, Novato, CA) was used to measure intracellular pH (pHi). Tubules were incubated with the acetoxymethyl derivative of the pH-sensitive dye BCECF (BCECF-AM; 5 × 10−6 M; Molecular Probes, Eugene, OR), which is permeable across cell membranes (1, 4, 12, 26). Intracellular esterases cleave the acetoxymethyl group, trapping BCECF in cells. A nigericin calibration curve was used to determine the pHi from the ratio of fluorescence (F500/F450), as described previously (1, 4, 12). Proximal convoluted tubules were then bathed with solution A that contained 1 mM SITS to inhibit the sodium bicarbonate cotransporter, the major regulator of proximal tubular pHi (1). This solution had 5 mM bicarbonate (pH 6.6) to compensate for the cell alkalinization caused by the addition of SITS to the bathing solution (2, 4, 21). After steady-state pHi was reached, the luminal perfusate was changed to one without sodium (solution C), and the rate of change in pHi was measured (dpHi/dt) to assess Na+/H+ exchange activity (1, 12, 13).

View this table:
Table 1.

Solutions

In vivo metabolic acidosis.

In some adult NHE3+/+/NHE8+/+ mice, we induced metabolic acidosis to examine whether both NHE3 and NHE8 were upregulated by acidosis in adults in vivo. Briefly, we placed adult mice on 0.28 M ammonium chloride for 1 wk in 2% sucrose (to improve palatability) and in addition gavaged the mice daily for the last 4 days before the study with 0.033 mmol/g body wt NH4Cl/g body wt (17, 30). Controls were given equivalent amounts of NaCl in their drinking water or by gavage.

Blood pressure.

Tail-cuff blood pressure was measured using a Visitech System BP-2000 Series II (Apex, NC) blood pressure monitor. Mice were placed in the holder, and blood pressure was measured several times daily for 4 days to train the mice. On day 5, actual readings were obtained. The mean of at least 10 readings was used as the reading for a mouse.

Statistical analysis.

Data are expressed as means ± SE. When two groups were compared, a Student's t-test was used. When there were more than two groups, analysis of variance with a post hoc Student-Neuman-Keuls test was used. A P value <0.05 was used to determine statistical significance.

RESULTS

NHE8−/− mice were generated by replacing 26 nucleotides in exon 8 with the LacZ-Neo gene as shown in Fig. 1. The result yielded a mouse where NHE8 mRNA was not transcribed. This is shown in Fig. 1 by the absence of cDNA using primers that span the insertion site and the absence of cDNA using primers 3′ from the insertion site. We also performed a RNA blot where we show the presence of NHE8 mRNA in the control mice but the complete absence in the NHE8−/− mice. Finally, Fig. 2 shows the absence of NHE3 and NHE8 protein in mice null for these transporters.

Fig. 1.

Generation and validation of Na/H exchanger 8 knockout (NHE8−/−) mice. A: gene deletion strategy. NHE8 mice were generated by replacing 26 nucleotides in exon 8 with the LacZ-Neo gene. B: mice were genotyped as shown. C: absence of cDNA using primers that span the insertion site of the LacZ-Neo gene D: absence of cDNA using primers after the insertion site. E: RNA blot showing the presence of NHE8 mRNA in the control mice but the complete absence in the NHE8−/− mice.

Fig. 2.

Immunoblot of NHE3 and NHE8 in wild-type and null mice. Immuoblots showing the presence and absence of NHE3 and NHE8 in wild-type and NHE null mice. Fifty micrograms of protein was loaded in each lane.

We first compared the blood pH and bicarbonate levels in NHE3+/+/NHE8+/+ (control), NHE3+/+/NHE8−/−, NHE3−/−/NHE8+/+, and NHE3−/−/NHE8−/− mice. As shown in Table 2, the blood pH and bicarbonate concentration is comparable in NHE3+/+/NHE8+/+ and NHE3+/+/NHE8−/− mice. NHE3−/−/NHE8+/+ mice had metabolic acidosis as previously described (23), which was not more severe in NHE3−/−/NHE8−/− mice. These data indicate that NHE3 and not NHE8 is the main determinant of plasma pH and bicarbonate levels at baseline. The serum sodium was slightly less in NHE3−/−/NHE8−/− mice and NHE3−/− mice than that of NHE3+/+/NHE8+/+ and NHE3+/+/NHE8−/− mice, which is likely secondary to the diarrhea that these mice have due to the importance of the Na+/H+ exchanger for sodium absorption in the intestine (23).

View this table:
Table 2.

Blood gas and electrolytes in neonatal control and NHE8−/− mice

We next examined whether there was a compensatory increase in NHE8 in NHE3−/− mice. We examined males and females separately since a recent study showed that NHE8 mRNA and protein expression was greater in adult female than adult male NHE3−/− mice intestines, which also normally express both NHE3 and NHE8 (35). As shown in Fig. 3, NHE8 mRNA abundance was highest in control males compared with NHE3−/− males and control and NHE3−/− females. In contrast to mRNA, NHE8 protein in both renal cortex and BBM was greater in NHE3−/− mice compared with control mice in both genders (Fig. 4, A and B). The increase in BBM NHE8 protein abundance in NHE3−/− mice was conceivably due to metabolic acidosis (see below).

Fig. 3.

NHE8 mRNA abundance in male and female control and NHE3−/− mice. RNA from mouse renal cortex were quantified by real-time RT-PCR and normalized to 28S rRNA. There were 7 mice in each group. Values are means ± SE. *P < 0.01 vs. other groups.

Fig. 4.

Male and female NHE8 total protein abundance in adult NHE3−/− and control mice. NHE3 protein was quantified by immunoblot and normalized to β-actin in renal cortex (A) and cortical brush-border membrane (BBM) vesicles (B). There were at least 4 mice in each group. Fifty micrograms of protein was loaded in each lane. Values are means ± SE. Some of the NHE8 bands are doublets due to protein glycosylation. *P < 0.05 vs. control of same sex.

We next determined whether NHE3 expression is modified in NHE8−/− mice. As shown in Fig. 5, NHE3 mRNA and total protein abundance was comparable in NHE8−/− and control mice. Despite the absence of metabolic acidosis in NHE8−/− mice, BBM NHE3 expression was greater in NHE8−/− mice without a detectable increase in total cortical NHE3 protein or mRNA. These findings are consistent with the model of NHE3 potentially playing a compensatory role in the absence of NHE8.

Fig. 5.

NHE3 Expression in NHE8−/− mice. NHE3 mRNA (A), total cortical protein (B), and BBM protein (C) were compared between control and NHE8−/− mice. Fifty micrograms of protein was loaded in each lane. There were 5 mice in each group. Values are means ± SE. *P < 0.05 compared with control.

We also examined whether NHE8 could play a role in the response to metabolic acidosis using wild-type adult mice. Mice given NH4Cl develop metabolic acidosis with a lower blood pH (7.13 ± 0.01 vs. 7.35 ± 0.01, P < 0.001) and bicarbonate concentration (13.2 ± 0.4 vs. 21.5 ± 0.3 mM, P < 0.001) than controls. Figure 6 demonstrates that both NHE3 and NHE8 protein are increased in BBM with metabolic acidosis. Thus NHE8, which is traditionally thought to be a developmental NHE isoform (7, 14, 28), is still expressed in adults and there is increased BBM expression in response to metabolic acidosis.

Fig. 6.

Effect of acidosis on BBM NHE3 and NHE8 protein abundance. Adult control mice were gavaged with acid or NaCl and renal BBM NHE8 (A) and NHE3 (B) were assayed by immunoblot. Fifty micrograms of protein was loaded in each lane. There were 6 mice in each group. Values are means ± SE. *P < 0.05.

To determine the relative functional importance of the two Na+/H+ exchangers, we measured Na+/H+ activity in NHE3+/+/NHE8+/+, NHE3+/+/NHE8−/−, NHE3−/−/NHE8+/+, and NHE3−/−/NHE8−/− mice. The baseline pHi in values in the presence and absence of luminal sodium are shown in Table 3. As shown in Fig. 7 there was no difference in proximal tubule Na+/H+ exchange in adult NHE3+/+/NHE8+/+ and NHE3+/+/NHE8−/− mice. However, NHE3−/−/NHE8+/+ mice had less Na+/H+ exchange activity than that of control mice as we have previously demonstrated (12). Interestingly, NHE3−/−/NHE8−/− mice had less Na+/H+ exchange activity than all other groups, including NHE3−/−/NHE8+/+ mice. Thus NHE8 plays a role in mediating Na+/H+ exchange in NHE3−/− mice.

View this table:
Table 3.

Intracellular pH (pHi) measurements in control, NHE8−/−, NHE3−/−, and NHE3−/−/NHE8−/− mice

Fig. 7.

Proximal tubule Na+/H+ exchange activity in NHE3+/+/NHE8+/+, NHE3+/+/NHE8−/−, NHE3−/−/NHE8+/+, and NHE3−/−/NHE8−/− mice. Murine proximal tubules were perfused in vitro, and intracellular pH (pHi) was measured by pH-sensitive fluorescence. NHE activity was measured by rate of change in pHi (dpHi/dt) in response to luminal Na removal. There were at least 10 tubules from at least 5 mice in each group. Values are means ± SE. *P < 0.01 vs. NHE3+/+/NHE8+/+ and NHE3+/+/NHE8−/− mice. #P < 0.05 vs. all other groups.

In the final series of experiments, we measured blood pressure in NHE3+/+/NHE8+/+, NHE3+/+/NHE8−/−, NHE3−/−/NHE8+/+, and NHE3−/−/NHE8−/− mice. These results are shown in Fig. 8. Blood pressure was comparable in NHE3+/+/NHE8+/+ and NHE3+/+/NHE8−/− mice. As previously shown, NHE3−/− mice have lower blood pressure than control mice (23). Interestingly, blood pressure in NHE3−/−/NHE8−/− mice was less than in all other groups, including NHE3−/− mice. Thus the absence of NHE8 does not affect blood pressure when NHE3 is intact; however, NHE8 may play a homeostatic role in preventing hypotension in the absence of NHE3.

Fig. 8.

Blood pressure in NHE3+/+/NHE8+/+, NHE3+/+/NHE8−/−, NHE3−/−/NHE8+/+, and NHE3−/−/NHE8−/− mice. Systolic blood pressure was measured by tail cuff in awake mice. The mean of at least 10 readings was used as the reading for a mouse. There were at least 6 mice in each group. Values are means ± SE. *P < 0.01 vs. NHE3+/+/NHE8+/+ and NHE3+/+/NHE8−/− mice. #P < 0.05 vs. all other groups.

DISCUSSION

Na+/H+ exchange plays a critical role in sodium chloride and bicarbonate absorption (5, 19, 2123, 31, 3133). While NHE3 is the predominant Na+/H+ exchanger in the adult proximal tubule, previous studies have demonstrated that there is residual apical membrane Na+/H+ exchange activity in NHE3−/− mice (12), which may be due to NHE8 (7, 15, 16). This study examined the relative contribution of NHE8 and NHE3 using NHE8−/− and NHE3−/− adult mice. NHE8−/− mice do not have metabolic acidosis nor a decrease in Na+/H+ exchanger activity compared with control mice, confirming that NHE3 is the predominant Na+/H+ exchanger in adult mice. However, there is a compensatory increase in BBM NHE3 protein abundance in NHE8−/− mice, and metabolic acidosis increases NHE8 protein abundance. We also demonstrate that NHE3−/−/NHE8−/− mice do not have more severe metabolic acidosis than NHE3−/− mice, arguing against a significant physiological role for NHE8 in adult mice at baseline. However, NHE3−/−/NHE8−/− mice have lower Na+/H+ exchanger activity and lower blood pressure than NHE3−/− mice, indicating that NHE8 may play a role in renal acidification to mediate Na+/H+ exchange activity in the absence of NHE3.

The segmental division of the nephron often displays redundant mechanisms for the reabsorption of solutes (25, 27, 28). Previous studies have examined whether there is renal compensation for the deletion of a transporter by increased expression of other transporters in the same or another nephron segment (11, 25). Of the three Na-coupled phosphate transporters in the proximal tubule BBM, NaPi-2a plays the predominant role in adult rodents while NaPi-2c is highly expressed in weaning rodents and has relatively low expression in adult rodents (24). The relative importance of these transporters has been clarified using NaPi-2a−/− and NaPi-2c−/− as well as double knockout mice (25). NaPi-2a−/− mice have hypophosphatemia and a higher fractional excretion of phosphate (6, 25), while the serum phosphorus and fractional excretion of phosphorus are normal in adult NaPi-2c−/− mice (25), creating the impression that NaPi-2c plays a minor or no role in phosphate transport in the adult mouse. However, NaPi-2a/NaPi-2c double knockout mice have a lower serum phosphorus level and higher fractional excretion of phosphate than NaPi-2a−/− mice, indicating that NaPi-2c is likely playing a compensatory role in NaPi-2a−/− mice (25). Similarly, when the renal-specific B1 subunit of the V-ATPase is deleted, B2 expression is increased to compensate and thus restore normal acid-base parameters (20).

We examined the potential compensatory role for NHE8 and NHE3 by measuring blood pH and bicarbonate levels in control, NHE3−/−, NHE8−/−, and NHE3−/−/NHE8−/− mice. NHE8−/− mice had a comparable blood pH and bicarbonate level as that of control mice. Metabolic acidosis as previously shown in NHE3−/− mice (23) was not more severe than in the NHE3−/−/NHE8−/− mice. These results are consistent with NHE3 playing the major role in renal acidification and NHE8 not having a significant role under basal conditions. We also examined whether there was an increase in mRNA and protein expression of NHE8 and NHE3 in NHE3−/− and NHE8−/− mice, respectively. We found an increase in NHE8 mRNA abundance in male but not in female NHE3 null mice. The reason for the higher expression in males is unclear. We find no effect of testosterone on NHE8 mRNA expression in NRK cells that express NHE8 but not NHE3 (data not shown). The changes in NHE8 mRNA were not mirrored by parallel changes in protein. We find that there is an equivalent increase in renal cortical and BBM NHE8 protein abundance in both male and female NHE3−/− adult mice compared with control littermates. Thus NHE8 is likely the Na+/H+ exchanger previously found in adult NHE3−/− mice (12). Unlike in the intestine, where postnatal females have higher NHE8 expression than males and only female NHE3−/− mice have increased expression of NHE8 compared with controls, we found comparable expression in male and female NHE3−/− mice, which was higher in both than in controls (35). Conversely, there was also an increase in NHE3 expression in NHE8−/− mice only in the BBM. This occurred despite the fact that there was no difference in serum pH, bicarbonate concentration, or Na+/H+ exchanger activity between NHE8−/− and control mice. This increase in NHE3 may be secondary to increased intestinal sodium loss in NHE8 null mice and not due to a direct renal compensation for the absence of NHE8. The factors that increase NHE3 in NHE8−/− mice are unclear and will need further study.

Na+/H+ exchanger activity was assayed in perfused tubules in the four groups of mice. We confirmed our previous findings that NHE3−/− mice had a lower rate of Na+/H+ exchanger activity compared with control mice (12). Nonetheless, there was significant EIPA-sensitive Na+/H+ exchanger activity present in NHE3−/− mice. NHE8−/− mice had comparable Na+/H+ exchanger activity to that of controls, indicating that under basal conditions, NHE8 does not play a significant role in adult renal acidification. NHE3−/−/NHE8−/− mice had less sodium-dependent H+ secretion than NHE3−/− mice. The transporter(s) responsible for the minimal residual sodium-dependent H+ secretion is unclear. These data are consistent with NHE8 being the predominant Na+/H+ exchanger previously hypothesized to be present in NHE3−/− mice (12). However, it should be appreciated that not all data point to a role for another Na+/H+ exchanger in NHE3−/− mice (31). In vivo proximal tubule microperfusion studies have shown that wild-type mice have an EIPA-inhibitable component of bicarbonate absorption, while NHE3−/− mice have a lower rate of bicarbonate absorption than wild-type mice that is not inhibited by EIPA (31). These data are not consistent with another EIPA-sensitive Na+/H+ exchanger compensating for the absence of NHE3. Previous studies have shown that NHE3−/− mice have lower blood pressure than control mice, which was found in this study as well (12). This is likely due to significant intestinal fluid losses as well as a decrease in sodium chloride transport by the proximal tubule (23, 31, 32).

In the present study, we found that NHE8−/− mice had comparable blood pressures to that of control mice. Interestingly, blood pressure of NHE3−/−/NHE8−/− mice was less than in all other groups, including NHE3−/− mice. This may be due to NHE8 having a compensatory role to maintain hemodynamics in the absence of NHE3. Since NHE8 is also expressed in the intestine (34, 35), it may be that NHE3−/−/NHE8−/− mice simply have more severe diarrhea than NHE3−/− mice, resulting in greater volume depletion and more severe hypotension It is also possible that homeostatic mechanisms acting on the Na+/H+ exchangers are not operative in the absence of both NHE8 and NHE3. We have previously examined BBM protein expression of NHE3 and NHE8 during postnatal development in mice and rats (7, 28). NHE3 protein abundance in the BBM increased with postnatal maturation, while NHE8 protein abundance in the BBM decreased with age. Renal BBM NHE8 is about four- to fivefold more highly expressed in 7- and 14-day-old mice compared with adult mice (28). It is not clear what the relative roles of NHE3 and NHE8 have in terms of renal acidification and whether NHE3 will compensate for the absence of NHE8 in neonatal NHE8−/− mice.

In conclusion, NHE3 is the predominant Na+/H+ exchanger. Adult proximal tubule acidification and deletion of NHE8 lead to no significant physiological changes in renal acidification or blood pressure homeostasis in adult mice. However, BBM NHE8 protein abundance increases with metabolic acidosis and may play a role to compensate for the lack of NHE3 in NHE3−/− mice.

GRANTS

This work was supported by National Institutes of Health Grants DK41612 to M. Baum and O. W. Moe, DK078596 to M. Baum, T32 DK07257 to Peter Igarashi and M. Baum, and the O'Brien Center P30DK079328.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: M.B., K.T., J.G., C.J., and O.W.M. provided conception and design of research; M.B., K.T., J.G., C.J., L.W., Q.Z., and V.D. performed experiments; M.B., K.T., J.G., C.J., L.W., Q.Z., and V.D. interpreted results of experiments; M.B., K.T., J.G., and C.J. prepared figures; M.B. and J.G. drafted manuscript; M.B., K.T., J.G., and O.W.M. edited and revised manuscript; M.B., K.T., J.G., C.J., L.W., Q.Z., V.D., and O.W.M. approved final version of manuscript; K.T., J.G., C.J., L.W., Q.Z., V.D., and O.W.M. analyzed data.

ACKNOWLEDGMENTS

We thank Dr. Gary Shull for the NHE3−/− mice.

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View Abstract