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Mouse model of type II Bartter's syndrome. II. Altered expression of renal sodium- and water-transporting proteins

¹Institute of Physiology, Center of Integrative Human Physiology, and ²Institute of Anatomy, University of Zurich, Zurich; ³Department of Medicine, Unit of Anatomy, University of Fribourg, Fribourg, Switzerland; and Departments of ⁴Cellular and Molecular Physiology and ⁵Surgery, Yale Medical School, New Haven, Connecticut

Submitted 25 December 2007 ; accepted in final form 28 February 2008

	ABSTRACT

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Bartter's syndrome represents a group of hereditary salt- and water-losing renal tubulopathies caused by loss-of-function mutations in proteins mediating or regulating salt transport in the thick ascending limb (TAL) of Henle's loop. Mutations in the ROMK channel cause type II antenatal Bartter's syndrome that presents with maternal polyhydramnios and postnatal life-threatening volume depletion. We have developed a colony of Romk null mice showing a Bartter-like phenotype and with increased survival to adulthood, suggesting the activation of compensatory mechanisms. To test the hypothesis that upregulation of Na⁺-transporting proteins in segments distal to the TAL contributes to compensation, we studied expression of salt-transporting proteins in ROMK-deficient (Romk^–/–) mice. Plasma aldosterone was 40% higher and urinary PGE₂ excretion was 1.5-fold higher in Romk^–/– compared with wild-type littermates. Semiquantitative immunoblotting of kidney homogenates revealed decreased abundances of proximal tubule Na⁺/H⁺ exchanger (NHE3) and Na⁺-P_i cotransporter (NaPi-IIa) and TAL-specific Na⁺-K⁺-2Cl^–-cotransporter (NKCC2/BSC1) in Romk^–/– mice, while the distal convoluted tubule (DCT)-specific Na⁺-Cl^– cotransporter (NCC/TSC) was markedly increased. The abundance of the -,β-, and -subunits of the epithelial Na⁺ channel (ENaC) was slightly increased, although only differences for -ENaC reached statistical significance. Morphometry revealed a fourfold increase in the fractional volume of DCT but not of connecting tubule (CNT) and collecting duct (CCD). Consistently, CNT and CD of Romk^–/– mice revealed no apparent increase in the luminal abundance of the ENaC compared with those of wild-type mice. These data suggest that the loss of ROMK-dependent Na⁺ absorption in the TAL is compensated predominately by upregulation of Na⁺ transport in downstream DCT cells. These adaptive changes in Romk^–/– mice may help to limit renal Na⁺ loss, and thereby, contribute to survival of these mice.

thick ascending limb of Henle's loop; distal convoluted tubule; thiazide-sensitive NaCl cotransporter; renal outer medulla K⁺ channel; hyperprostaglandin E syndrome

Impaired Na⁺ transport in the TAL leads to enhanced downstream Na⁺ delivery to the DCT, connecting tubule (CNT), and cortical collecting duct (CCD) (for a review, see Refs. 16, 18, and 25). Previous studies in rats have shown that chronic inhibition of sodium absorption in the TAL by loop diuretics like furosemide causes hypertrophy and hyperplasia of the DCT, CNT, and CCD associated with an increased abundance of the apical Na⁺-Cl^– cotransporter (NCC), epithelial Na⁺ channel (ENaC), and basolateral Na⁺-K⁺-ATPase (1, 9, 14, 17, 24, 28). These adaptive mechanisms have been suggested to play a role in the development of furosemide resistance that occurs frequently during treatment with loop diuretics (25). Similar functional adaptations of the distal nephron could help compensate for derangements of function of the loop of Henle in HPS. Thus, in the present study, we examined the adaptive responses in ion transport proteins along the nephron in the ROMK null mouse model of type II Bartter's syndrome.

	MATERIALS AND METHODS

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Mice. The generation of Romk^+/+ and Romk^–/– mice has been previously described (21; see also Ref. 4). Survival of Romk^–/– mice to adults was >50%. Wild-type Romk^+/+ are genetically identical to Romk^–/– mice except for deletion of Kcnj1. All mice were given tap water ad libitum and maintained on standard rodent chow (1.2% K). All animal experiments were approved by the Yale Animal Care Committee.

Urine collection. Mice were housed in metabolic cages with free access to normal food and water. Urine samples were collected over 16 h into tubes immersed in ice during collection.

Serum PGE₂ and aldosterone measurements. PGE₂ concentrations were determined by using a PGE₂ EIA Kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. Briefly, after dilution of urine samples to the optional concentration, 50 µl of each sample was mixed along with a serial dilution of PGE₂ standard samples with appropriate amounts of acetylcholinesterase-labeled tracer and PGE₂ antiserum and incubated at 4°C for 18 h. After the wells were emptied and rinsed with wash buffer, 200 µl of Ellman's reagent containing the acetylcholinesterase substrate was added. The enzyme reaction was carried out on a slow shaker at room temperature for 1–2 h. The plates were read on a Microplate Reader (Benchmark Microplate Reader, Bio-Rad) at 405 nm. The results were analyzed with Cayman Chemical's computer spreadsheet.

Plasma aldosterone concentrations were measured in blood samples collected from the carotid artery using an Immuchem Double Antibody, Aldosterone ¹²⁵I RIA kit at Yale University's General Clinical Research Center. The RIA kit was purchased from ICN Pharmaceuticals (Costa Mesa, CA).

Antibodies. The antibodies used for immunohistochemistry and Western blotting are shown in Table 1.

All sera were characterized by immunofluorescence and standard Western blotting with mouse kidney samples. Specific binding of the antibodies to kidney sections (immunofluorescence) and to polyvinylidene difluoride (PVDF) membranes (Western blotting) could be completely inhibited by preincubation of the antisera with the specific peptides used for immunization (see supplementary figures; all supplementary material is available in the online version of this article at the journal web site).

Western blotting. The mice were killed, and the kidneys were rapidly harvested. Total kidneys were homogenized in an ice-cold K-HEPES buffer (200 mM mannitol, 80 mM K-HEPES, 41 mM KOH, pH 7.5) with pepstatin, leupeptin, K-EDTA, and phenylmethylsulfonyl fluoride (PMSF) added as protease inhibitors. The samples were centrifuged at 1,000 g for 10 min at 4°C, and the supernatant was saved. Subsequently, the supernatant was centrifuged at 100,000 g for 1 h at 4°C, and the resultant pellet was resuspended in K-HEPES buffer containing protease inhibitors. After measurement of the total protein concentration (Bio-Rad Protein kit), 50 µg of crude membrane protein were solubilized in Laemmli sample buffer, and SDS-PAGE was performed on 10% polyacrylamide gels. For immunoblotting, proteins were transferred electrophoretically from unstained gels to PVDF membranes (Immobilon-P, Millipore, Bedford, MA). After blocking with 5% milk powder in Tris-buffered saline/0.1% Tween 20 for 60 min, the blots were incubated with the primary antibody either for 2 h at room temperature or overnight at 4°C. After washing and subsequent blocking, blots were incubated with the secondary antibodies (donkey anti-rabbit 1:10,000 and sheep anti-mouse 1:5,000, IgG conjugated with horseradish peroxidase, Amersham Life Sciences) for 1 h at room temperature. Antibody binding was detected with an enhanced chemiluminescence ECL kit (Amersham Pharmacia Biotech) before exposure to X-ray film (Kodak). The protein/actin ratio was determined and used to calculate the ratio between the wild-type and ROMK-deficient group using Gauss' law of error propagation. All results were tested for significance using the unpaired Student's t-test, and only results with P < 0.05 were considered as statistically significant.

Western blot analysis of renin expression. Mouse kidneys were homogenized in 1–2 ml of extraction buffer (5 mM Tris hydrochloride, pH 7.5, 2 mM EDTA, 1% SDS, 100 µM PMSF, 2 µg/ml leupeptin, and 2 µg/ml aprotinin), and centrifuged at 4,000 g for 30 min at 4°C. Protein concentration was determined using a BCA Protein assay kit (Pierce, Rockford, IL). Forty micrograms of protein was solubilized in 2x sample buffer, separated by 10% SDS-PAGE, and transferred to PVDF membranes using the Mini-Trans blotting system (Bio-Rad Laboratories, Hercules, CA). Prestained high-range SDS-PAGE standards were used (Bio-Rad Laboratories). The membranes were blocked for 1 h in 5% nonfat milk/TBS-Tween at room temperature before incubation with the primary mouse monoclonal anti-renin antibody (Research Diagnostics) diluted 1:1,000 in 5% nonfat milk/TBS-Tween overnight at 4°C. After washing, membranes were incubated with peroxidase-linked anti-mouse IgG antibodies (Amersham Life Science, Arlington Heights, IL) for 1 h at room temperature. After washing in TBS-Tween, antigen-antibody complexes were detected using the ECL chemiluminescence kit (ECL, Amersham Life Science).

Immunohistochemistry. Mice were anesthetized with ketamine/xylazine (ip). and perfused through the left ventricle with PBS followed by paraformaldehyde-lysine-periodate fixative (23). Immunohistochemistry was performed as previously described (20). Briefly, cryosections 5 µm thick were incubated overnight at 4°C with antibodies given in Table 1. Binding sites were revealed by Cy3-conjugated goat anti-rabbit IgG antibodies. Sections were studied by epifluorescence on a Zeiss microscope. Histological and morphometrical analysis was performed by two experienced investigators blinded for the genotype of the animals. Digital images were taken with a charge-coupled device camera.

Quantification of -ENaC by real-time PCR. ROMK wild-type and knockout mice were killed by injection of ketamine/xylazine (ip) and subsequent cervical dislocation, and the kidneys were collected and rapidly frozen until further use. Total mRNA was extracted from 30 mg of tissue using an RNA Aqueous 4PCR kit (Ambion) according to the manufacturer's instruction. For RNA extraction, kidneys was thawed in RNALater solution (Ambion), transferred to lysis buffer, and homogenized on ice with an Elvehjem potter. RNA was bound on columns and treated with DNAse for 15 min at 30°C to reduce genomic DNA contamination. Quantity and purity of total eluted RNA were assessed by spectrometry and on agarose gels. Each RNA sample was diluted to 200 ng/µl, and 3 µl was used as a template for reverse transcription using a Taqman Reverse Transcription kit (Applied Biosystems) in the presence of 2.5 µM Random Hexamers primers (Applied Biosystems).

For reverse transcription, a 200-ng RNA template was diluted in a 20-µl reaction mix that contained (final concentrations) RT buffer (1x), MgCl₂ (5.5 mM), random hexamers (2.5 µM), RNAse inhibitor (0.4 U/µl), the multiscribe RT enzyme (1.25 U/µl), deoxyNTP mix (500 µM each), and RNAse-free water. Real-time PCR was performed as described previously, according to the recommendations supplied by Applied Biosystems (http://home.appliedbiosystems.com). Primers for all genes of interest were designed using Primer Express 2.0 software from Applied Biosystems. Primers were chosen to result in amplicons of 70–100 bp that span intron-exon boundaries. The specificity of all primers was first tested on mRNA derived from kidneys and resulted in a single product of the expected size (data not shown). Probes were labeled with the reporter dye FAM at the 5'-end and the quencher dye TAMRA at the 3'-end (Microsynth, Balgach, Switzerland). The passive reference dye (ROX) was included in the Taqman buffer supplied by the manufacturer. Twenty microliters of cDNA obtained from the RT reaction was diluted to 100 µl with RNAse-free water. A 25-µl PCR reaction volume was prepared using 5 µl diluted cDNA as a template with sense and antisense primers (25 µM each) and the labeled probe (5 µM). The sequences for primers used were the following: β-actin forward primer: 5'-CCACCGATCCACACAGAGTACTT-3', β-actin reverse primer: 5'-GACAGGATGCAGAAGGAGATTACTG-3'; and -ENaC forward primer: 5'-GGTGCACGGTCAGGATGAG-3', -ENaC reverse primer: 5'-TAGTTGCCTCCGAGGCTGTC-3'. The Taqman Universal PCR Master Mix (Applied Biosystems) was added to the final volume. Reactions were run in 96-well optical reaction plates using a Prism 7700 cycler (Applied Biosystems).

Thermal cycles were set at 95°C (10 min) and the 40 cycles at 95 (15 s) and 60°C (1 min) with auto-ramp time. To analyze the data, the threshold was set to 0.06 (value in the linear range of amplification curves). All the reactions were run in triplicate. The abundance of -ENaC mRNA was calculated relative to a reference mRNA (mouse β-actin). Assuming an efficiency value of 2 (fold-increase in input mRNA required to decrease the cycle number by 1), relative expression ratios were calculated as

where C_t is the cycle number at the threshold.

In situ microperfusion of mouse kidney proximal tubules. The net fluid and HCO₃^– absorption in proximal tubules of age-matched adult ROMK null and wild-type mice was assessed by in situ microperfusion as described previously (32). Briefly, superficial proximal convoluted tubules were perfused at a rate of 15 nl/min with a solution containing 115 mM NaCl, 25 mM NaHCO₃, 4 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 2 mM NaPO₄, and 20 µCi/ml low-Na⁺ methoxy-[³H]inulin (pH 7.4). The rates of net fluid (J_v) and HCO₃^– (J_HCO3) absorption were calculated based on changes in the concentrations of [³H]inulin and total CO₂ as described previously (33). J_v and J_HCO3 are expressed per minute per millimeter of proximal tubule.

	RESULTS

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Hormone levels. HPS is associated with increased urinary excretion of PGE₂ (12). Measurement of urinary PGE₂ excretion in ROMK null mice demonstrated a nonsignificant 1.5-fold increase in PGE₂ levels in (PGE₂ wild-type: 1,099.9 ± 107.3 pg/day; Romk^–/–: 15,743.0 ± 209.2 pg/ day, n = 12, P = 0.056). Plasma aldosterone was slightly but significantly higher in Romk^–/– animals than in Romk^+/+ mice (260.3 ± 31.0 vs. 181.5 ± 11.5 pg/ml, P < 0.05). In agreement with previous reports (12, 26) on enhanced plasma renin activity in patients with Bartter's syndrome, we found by Western blotting and immunohistochemistry increased renin abundance in kidneys from Romk^–/– mice (data not shown).

Changes in proximal tubule salt and water transport proteins and transport rates in Romk^–/– mice. Figure 1 shows the absence of ROMK expression in kidneys of Romk^–/– mice by Western blotting of total crude membranes (Fig. 1A) and immunofluorescence staining of tissue sections (Fig. 1B). The abundance of the proximal tubule Na⁺-transport proteins Na⁺/H⁺ exchanger isoform 3 (NHE3) and Na⁺-phosphate cotransporter type IIa (NaPi-IIa) was reduced by 10 ± 6 and 37 ± 12%, respectively (Fig. 2) in kidneys of Romk^–/– mice compared with those of wild-type mice. This reduction may reflect the relative decrease in proximal tubular mass due to the increased fractional cortical tubular volume of distal convoluted tubules as described below. However, this 10% reduction of NHE3 expression in kidneys of Romk^–/– mice did not significantly influence net Na⁺ and HCO₃^– absorption in proximal tubules. As show in Fig. 3, J _v and J_HCO3 absorption were not significantly different between Romk^+/+ and Romk^–/– mice. Although the glomerular filtration rate in Romk^–/– mice was 50% of control Romk^+/+ mice (4), the single-nephron glomerular filtration rate was not reduced in Romk^–/– mice (21). Since NHE3 activity is flow dependent (6), the 10% reduction of NHE3 expression was not detectable by changes in J_v and J_HCO3 absorption at the flow rate close to the normal single-nephron glomerular filtration rate in the proximal tubule.

View larger version (81K): [in this window] [in a new window]   Fig. 1. Absence of ROMK from kidneys from ROMK-deficient mice (Romk–/–). A: semiquantitative immunoblotting with homogenates from total kidneys of wild-type (Romk+/+) and Romk–/– mice. Fifty micrograms of crude membrane preparations from each mouse were subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane and probed with affinity-purified anti-ROMK antibodies (Alamone Laboratories) and reprobed for β-actin after being stripped to control for loading. B: medullary rays in renal cortex of Romk+/+ and Romk–/– mice. Immunostaining for ROMK with affinity-purified rabbit-anti-ROMK antibody shows prominent ROMK staining in the luminal membrane of some thick ascending limb (T) cells and in the cytoplasm of some collecting duct (CD) cells that is absent from kidneys of ROMK-deficient mice. Scale bar = 50 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 2. Altered abundance of proximal tubular transport proteins in ROMK-deficient mice. Semiquantitative immunoblotting was performed with 50 µg of crude membrane preparations from Romk+/+ and Romk–/– mouse kidneys. Compared with wild-type mice, ROMK-deficient mice exhibit reduced renal abundance of Na+-Pi cotransporter type IIa (NaPi-IIa) and Na+/H+ exchanger isoform 3 (NHE3).

View larger version (7K): [in this window] [in a new window]   Fig. 3. Fluid and bicarbonate absorption in kidney proximal tubules The net fluid (Jv) and bicarbonate (JHCO3) absorption were measured by microperfusion of proximal tubules in situ in both Romk+/+ and Romk–/– mice.

View larger version (93K): [in this window] [in a new window]   Fig. 4. Reduced abundance and immunostaining for the thick ascending limb of Henle's loop (TAL)-specific Na+-K+-2Cl– cotransporter (NKCC2/BSC1). A: semiquantitative immunoblotting with 50 µg of crude membrane preparations from Romk+/+ and Romk–/– mouse kidneys. ROMK-deficient mice showed reduced abundance of the TAL-specific NKCC2. B: outer and inner stripe (OS and IS, respectively) of renal outer medulla of Romk+/+ and Romk–/– mice. Immunostaining for NKCC2 is seen in the luminal membrane of TAL in wild-type and ROMK-deficient mice. The epithelial cells of TALs in ROMK-deficient mice do not show any gross abnormalities but apparently reveal weaker NKCC2 immunofluorescence than the TALs of wild-type mice. Scale bar = 50 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 5. Compensatory increased abundance in the thiazide-sensitive Na+-Cl– cotransporter (NCC) and massive hypertrophy of the distal tubule in kidneys from ROMK-deficient mice. A: semiquantitative immunoblotting with 50 µg of crude membrane preparations from Romk+/+ and Romk–/– mouse kidneys. ROMK-deficient mice showed increased abundance of distal convoluted tubule (DCT)-specific NCC. B and C: overviews of renal cortex of Romk+/+ and Romk–/– mice. Immunofluorescence with rabbit anti-NCC antibodies reveals much higher density of NCC-positive DCT profiles in the renal cortex of the Romk–/– mouse than in the renal cortex of the Romk+/+ mouse. Scale bar = 200 µm. D and E: higher magnification of NCC-immunostained DCT profiles demonstrates the tremendous hypertrophy of the DCT epithelium in ROMK-deficient mice. Pictures were taken at the same magnification. Scale bar = 20 µm. F: fractional cortical volumes of DCT and the cortical collecting system [connecting tubule (CNT) and cortical collecting duct (CCD)] in Romk+/+ and Romk–/– mice. DCT and CNT/CCD were identified on account of their characteristic antibody-binding [DCT: NCC positive; CNT/CCD: NCC negative; and β-epithelial sodium channel (ENaC) positive] as described in MATERIALS AND METHODS. The fractional cortical tubular volumes were determined by the point-counting method, according to Weibel (35). Values are means ± SE from 4 individual mice in each group. *P < 0.05 vs. Romk–/– values.

View larger version (81K): [in this window] [in a new window]   Fig. 6. Abundance and localization of ENaC subunits. A: semiquantitative immunoblotting with 50 µg of total membrane preparations from kidneys of Romk+/+ and Romk–/– mice. The abundance of the -ENaC and -ENaC subunits is profoundly increased, whereas the expression of β-ENaC appears to be unaffected. Polyvinylidene difluoride (PVDF) membranes were stripped and reprobed for β-actin to ensure equal loading. B: relative abundance of mRNA for the aldosterone-regulated -ENaC as determined by real-time PCR and normalized against β-actin mRNA. C–F: consecutive cryosections of renal cortex of Romk+/+ (C and D) and Romk–/– (E and F) mice immunostained for NCC (C and E) and β-ENaC (D and F). Note the marked hypertrophy of early DCT (D) profiles, (characterized by strong NCC, but no β-ENaC immunoreactivity) in Romk–/– mice compared with early DCTs of Romk+/+ mice. The epithelial heights of late DCT profiles (asterisks), which are characterized by costaining for NCC and β-ENaC, and of CNT profiles (CN) do not apparently vary between the two mice. Similarly, mice of both genotypes show apical β-ENaC immunostaining only in the late DCT (asterisk) and in the directly adjacent early connecting tubule (CN). In farther downstream localized CNT (CN*) segments, β-ENaC is almost exclusively localized to intracellular compartments in mice of both genotypes. Scale bar = 40 µm. G and H: higher magnification of CNT profiles from Romk+/+ and Romk–/– mice demonstrate the predominant intracellular localization of -ENaC in the CNTs from mice of both genotypes. Unstained cells in the CNT epithelia represent intercalated cells, which are known to be ENaC negative. Scale bar = 40 µm.

View larger version (81K): [in this window] [in a new window]   Fig. 7. Expression and localization of the vasopressin-regulated aquaporin-2 (AQP2) water channel. A: semiquantitative immunoblotting for AQP2 with total membrane preparations from total kidney of Romk+/+ and Romk–/– animals reveals no significant difference between both genotypes. B–D: medullary rays in renal cortex of Romk+/+ and Romk–/– mice. Immunostaining for AQP2 with affinity-purified rabbit anti-AQP2 antibody. B: AQP2 immunofluorescence is visible in the luminal membrane and in the cytoplasm of collecting duct principal cells. Intercalated cells are AQP2 negative. The collecting ducts of ROMK-deficient mice often exhibit more pronounced intracellular and apical AQP2 immunostaining (C). In some collecting duct profiles of ROMK-deficient mice (D), AQP2 is almost exclusively localized at the apical plasma membrane. Scale bar = 20 µm.

	DISCUSSION

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These results are in agreement with functional data obtained in the same animals, as described in the companion study (4). The reduction of NKCC2 expression and a reduced effect of furosemide on the fractional Na⁺ excretion rate together demonstrate that ROMK is critical for TAL function. The absence of apparent structural changes in the TAL is consistent with previous morphological studies using kidneys of furosemide-treated rats (12, 13). Moreover, it appears that most of the lost TAL function is compensated for by the subsequent distal tubule as indicated by the massive hypertrophy of the DCT epithelium, the increase in the expression levels of the thiazide-sensitive NaCl-cotransporter NCC, and the exaggerated response in urinary Na⁺ excretion after application of thiazide diuretics. The fractional cortical tubular volume of the DCT increased >300%, which readily explains the previously reported higher volume density of distal tubules in Romk^–/– mice (21). The structural changes in the DCT are reminiscent of those seen in furosemide-treated rats. In these rats, the pharmacological inhibition of TAL function also induced DCT cell growth (13, 14), which was, however, less dramatic as that seen in Romk^–/– mice. Although not specifically addressed in the present study, results from furosemide-treated rats indicate that the observed adaptive DCT growth originates from both epithelial hypertrophy (9, 14) and epithelial hyperplasia (17). PGE₂ has been implicated in the hypertrophy of distal nephron segments but has also been made responsible, at least in part, for the pathophysiology observed in Bartter's syndrome (12). Hence, it remains to be clarified whether PGE₂ is part of the compensatory response or rather contributes to the observed renal defect. ROMK-deficient mice will provide an excellent model for the study of this problem in the future.

Although Romk^–/– mice have a severe Na⁺-transporting defect in the TAL, the animals are apparently more or less in sodium balance, as indicated by the only slightly elevated plasma renin and aldosterone levels. Consistent with the rather minor effect on plasma aldosterone levels, Romk^–/– mice did not show any significant upregulation of the aldosterone-dependent -ENaC subunit at the mRNA level and no effect on the cell surface localization of all three ENaC subunits. Similarly, the renal clearance experiments (see Ref. 4, the companion study) did not show any significant difference between the amiloride-sensitive portion of renal Na⁺ reabsorption in wild-type and ROMK-deficient mice. Together with the lack of any effect on the fractional cortical tubular volume of the collecting system, these data strongly suggest that ENaC-mediated Na⁺ transport in the collecting system is not significantly activated and therefore does not significantly contribute to the compensation of the renal Na⁺ transport defect in ROMK-deficient mice. This stands in sharp contrast to the compensatory mechanisms in NCC-deficient mice. These mice revealed elevated plasma aldosterone levels, increased ENaC cell surface abundance, and a marked epithelial hypertrophy of the renal connecting tubule but not of the collecting duct (20). Taken together, these findings suggest that the genetic salt transport defect in a given distal tubular segment is largely compensated by structural and functional adaptations in the directly following downstream tubular segment. Consistent with this hypothesis is our observation in the present study that the DCT hypertrophy in Romk^–/– mice is most pronounced in the early DCT.

Interestingly, no clear effect was found in AQP2 water channels. Despite the fact that Romk^–/– animals are polyuric and that some of them showed a more pronounced apical AQP2 staining with the appearance of AQP2 channels also in the CNT, this effect was not observed in all animals. Immunoblotting also showed no significant difference between wild-type and knockout animals.

In summary, ablation of ROMK channels in the TAL in the mouse kidney leads to loss of TAL function. Excessive water and salt loss are prevented by a combination of hypertrophy of the subsequent distal nephron and upregulation of NaCl reabsorption via the thiazide-sensitive NCC cotransporter and to a lesser extent via ENaC. Upregulation of AQP2 channels appears to play a minor role and was not observed in all animals. Thus the ROMK-deficient mouse model provides good evidence for compensatory mechanisms in Bartter's syndrome and TAL dysfunction and will allow study of the mechanism underlying these compensatory processes.

	GRANTS

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This study was supported by grants from the Swiss National Research Foundation to C. A. Wagner (31-109677/1) and J. Loffing (3200B0-105769), the EU 6th framework program EUREGENE to C. A. Wagner, a Marie Heim-Vögtlin fellowship and the Research Fund of the University of Fribourg to D. Loffing-Cueni, and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54999 (to S. C. Hebert) and DK-62289 and DK-17433 (to T. Wang).

	ACKNOWLEDGMENTS

 
We thank Drs. O. Moe, H. Murer, and B. Rossier for providing antibodies against NHE3, NaPi-IIa, and ENaC, respectively.

	FOOTNOTES

 

Addresses for reprint requests and other correspondence: C. A Wagner, Institute of Physiology, Center for Integrative Human Physiology, Univ. of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland (e-mail: Wagnerca{at}access.uzh.ch) and J. Loffing, Institute of Anatomy, Univ. of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland (e-mail: johannes.loffing{at}anatom.uzh.ch)

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.

^* C. A. Wagner and D. Loffing-Cueni contributed equally to this work.

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