Macula Densa Control of Secretion and Preglomerular Resistance in Mice with Selective Deletion of the B Isoform of the Na,K,2Cl Co-Transporter

Mona Oppermann,* Diane Mizel,* George Huang,* Cuiling Li,* Chuxia Deng,* Franziska Theilig,† Sebastian Bachmann,† Josie Briggs,* Jurgen Schnermann,* and Hayo Castrop*‡ *National Institute of Diabetes and Digestive and Diseases, National Institutes of Health, Bethesda, Maryland; †Anatomy, Charite, Humboldt University, Berlin, Germany; and ‡Institute of Physiology, University of Regensburg, Regensburg, Germany

Na,K,2Cl co-transporter (NKCC2), the primary NaCl uptake pathway in the thick ascending limb of Henle, is expressed in three different full-length splice variants, called NKCC2F, NKCC2A, and NKCC2B. These variants, derived by differential splicing of the variable exon 4, show a distinct distribution pattern along the , but the functional significance of this organization is unclear. By introduction of premature stop codons into exon 4B, specific for the B isoform, mice with an exclusive NKCC2B deficiency were generated. Relative expression levels and distribution patterns of NKCC2A and NKCC2F were not altered in the NKCC2B-deficient mice. NKCC2B-deficient mice did not display a salt-losing phenotype; basal plasma renin and aldosterone levels were not different from those of wild-type mice. Ambient urine osmolarities, however, were slightly but significantly reduced. Distal Cl concentration was significantly elevated and loop of Henle Cl absorption was reduced in microperfused superficial loops of Henle of NKCC2B-deficient mice. Because of the presence of NKCC2A in the macula densa, maximum tubuloglomerular feedback responses were normal, but tubuloglomerular feedback function curves were right-shifted, indicating reduced sensitivity in the subnormal flow range. Plasma renin concentration in NKCC2B-deficient mice was reduced under conditions of salt loading compared with that in wild-type mice. This study shows the feasibility of generating mice with specific deletions of single splice variants. The mild phenotype of mice that are deficient in the B isoform of NKCC2 indicates a limited role for NKCC2B for overall salt retrieval. Nevertheless, the high-affinity NKCC2B contributes to salt absorption and macula densa function in the low NaCl concentration range. J Am Soc Nephrol 17: 2143–2152, 2006. doi: 10.1681/ASN.2006040384

n addition to being affected by numerous systemic factors, densa cells is thought to be the mechanism by which these cells substantial evidence supports the concept that renal vas- detect tubular salt concentration (7–12). I cular resistance and renin secretion are the end points of In all mammalian species so far examined, NKCC2 is ex- an intrarenal regulatory pathway that resides in the juxtaglo- pressed along the thick ascending limb of Henle (TAL) in at merular apparatus. By connecting tubular epithelial and pre- least three different splice variants, called NKCC2F, NKCC2A, glomerular vascular cells, this structure provides an anatomic and NKCC2B (13–17). These variants are derived from one route along which information about composition distinct gene by differential splicing of the variable exon 4, can be signaled to vascular effector cells of the same . which encodes for the second transmembrane domain and Specifically, renin secretion and preglomerular resistance have parts of the adjacent intracellular loop of the transporter protein been shown to be strongly affected by changes of luminal NaCl (14,15,18). NKCC2F seems to be the dominant isoform of concentration at the level of the epithelial sensor cells of the NKCC2, with highest expression levels in the medullary por- macula densa (1–6). Transport activity of the Na,K,2Cl co- tion of the TAL (13,19). NKCC2A and NKCC2B seem to be transporter (NKCC2; BSC1) in the apical membrane of macula expressed in the more distal or cortical segments of the TAL (13,19). A recent attempt to localize NKCC2 isoforms by in situ hybridization in the mouse suggested that macula densa cells

Received April 21, 2006. Accepted May 18, 2006. seem to express exclusively and selectively the B isoform of the co-transporter (13). Therefore, NKCC2B seemed to be the most Published online ahead of print. Publication date available at www.jasn.org. likely candidate for serving as the salt sensor in macula densa H.C.’s current address is Institute of Physiology, University of Regensburg, control of renin secretion and preglomerular resistance. Germany. Studies of the NKCC2 sensor function in macula densa con- Address correspondence to: Dr. Hayo Castrop, NIDDK, NIH, Building 10, Room 4 D51, 10 Center Drive MSC-1370, Bethesda, MD 20892-1370; Phone: 301-435-6579; trol of renin secretion and preglomerular resistance in vivo Fax: 301-435-6587; E-mail: [email protected] traditionally have been performed by examination of the effects

Copyright © 2006 by the American Society of Nephrology ISSN: 1046-6673/1708-2143 2144 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 2143–2152, 2006 of loop diuretics such as furosemide or bumetanide (20,21). However, these agents block the transport activity of all iso- forms of NKCC2. Complete inactivation of NKCC2 by gene targeting results in severe electrolyte disturbances that lead to early postnatal death in most animals and therefore represents a model that cannot be studied easily physiologically (22). In these experiments, we assessed the effect of a more local- ized inactivation of NKCC2 on macula densa function. On the basis of the premise that NKCC2B is the dominant isoform of NKCC2 in macula densa cells, we generated a mouse strain that specifically is deficient in NKCC2B. NKCC2B-deficient mice showed an impairment of TAL diluting function accompanied by a slight decrease of osmotic urine concentration and a sig- nificant right shift of the tubuloglomerular feedback (TGF) function curve. Renin secretion was suppressed during salt loading. Partial maintenance of macula densa signaling indi- cates that another NKCC2 isoform in addition to NKCC2B must be involved in the macula densa sensing function.

Materials and Methods Generation of NKCC2B-Deficient Mice All animal studies were performed according to protocols that were examined and approved by the Animal Use and Care Committee of the National Institute of Diabetes and Digestive and Kidney Diseases and by the Animal Care Committee of the University of Regensburg. A targeting vector was generated to introduce premature stop codons into exon 4B of the NKCC2 gene (Slc12a1; Figure 1, A and C). This targeting vector consisted of a 5Ј homologous arm that was introduced into the vector ploxP (23), followed by the coding sequence for the FLAG peptide, by stop codons in all reading frames, and by a neomy- cin-resistance (neo) cassette flanked by loxP sites. 3Ј-Adjacent to the neo cassette, a 3Ј homologous arm was inserted (Figure 1B). Homologous arms were generated by long-distance PCR (Roche, Indianapolis, IN). Primers used were 5Ј-GGAAGGTTATTTGGGCTTGGTCCTG-3Ј and 5Ј-ACACCGAGACCTCAGTTGAGAGG-3Ј for the upstream arm and 5Ј-GATTATCATCGGCTTAGCCGTGACAG-3Ј and 5Ј-GGAGTTGC- CACACTACATGGGCTC-3Ј for the downstream arm. The targeting vector was linearized with NotI and transfected into 129SvEv embry- Figure 1. (A) Differential splicing of the variable exon 4 results onic stem cells. Primers used for PCR screening of the clones were in Na,K,2Cl co-transporter B (NKCC2B), NKCC2A, and 5Ј-CGCAGCGCATCGCCTTCTATCGCCTTC-3Ј (inside the neo cas- NKCC2F transcripts. (B) Targeting strategy in which introduc- sette) and 5Ј-CCACGGTGATGGAACCGATGATG-3Ј (3Ј outside of the tion of premature stop codons into exon 4B leads to premature downstream homologous region). Embryonic stem cell clones that had stop of translation; the vector also contained the FLAG peptide undergone homologous recombination were injected into blastocysts of coding sequence upstream of the stop codons (not shown in the C57BL/6 mice and implanted into pseudopregnant foster female mice. scheme). Arrows indicate PCR-based screening strategy for Two male chimeras were crossed to C57BL/6 female mice to test for alleles that had undergone homologous recombination. (C) germline transmission. Genotyping was performed on tail biopsies by Schematic drawing of the NKCC2 protein with arrow indicat- PCR using primers for the neo gene (5Ј-ACAACAGACAATCGGCT- ing location of translational stop. GCTCTGATG-3Ј and 5Ј-TGCGCGCCTTGAGCCTGGCGAAC-3Ј) and primers flanking the wild-type or the mutated exon 4B, respectively (5Ј-CGAGGGCAACAGAGCTCTGCACATTCC-3Ј and 5Ј-GGTCAGT- Analysis of the Mutated Transcripts of NKCC2BϪ/Ϫ Mice Ј GGTGAGCTATTGGATGGAGC-3 ). Total RNA from kidneys of NKCC2Bϩ/ϩ and Ϫ/Ϫ mice was isolated using Trizol reagent (Life Technologies, Carlsbad, CA). NKCC2B mutated transcripts were analyzed by reverse transcrip- Removal of the Neo Cassette tion–PCR (RT-PCR) using primers that spanned from exon 4B to The loxP-flanked neo cassette was excised by crossing homozygous exon 5 and from exon 2 to exon 4B. To verify the integrity of the NKCC2BϪ/Ϫneoϩ mice with EIIa-Cre mice (24). Offspring DNA was remaining isoforms, we also performed analogous RT-PCR for tested for the absence of neo, and progeny that lacked the neo gene NKCC2A and NKCC2F transcripts. In addition, cDNA fragments were intercrossed to obtain homozygous NKCC2BϪ/Ϫ mice and wild- that spanned from exon 2 to exon 5 were cloned into TOPO XL (Life type littermates (Figure 1B). Technologies), and all fragments were sequenced. J Am Soc Nephrol 17: 2143–2152, 2006 NKCC2B Deletion and Kidney Function 2145

Localization of NKCC2B, NKCC2A, and NKCC2F For determination of absorptive TAL function, loops of Henle were Transcripts in Kidney Segments perfused as described above. Fluid samples were collected from distal A localization study for the three different isoforms of NKCC2 was tubular segments of the perfused segments for 1 to 3 min at set flow performed by real-time RT-PCR on RNA that was derived from differ- rates of 6 and 15 nl/min. After volume determinations, a 1-nl sample Ϫ ent kidney regions. Kidneys were dissected under a microscope, and was removed for determination of Cl concentration using an electro- Ϫ cortex, outer medulla, and inner medulla tissue pieces were collected. metric Cl microtitrator (28). Real-time PCR was performed using isoform-specific TaqMan probes in an ABI Prism 7900 Sequence Detection System (Applied Biosystems, Statistical Analyses Foster City, CA). Unpaired t test was used to compare two values between different animals. Multiple groups were analyzed with ANOVA followed by Localization of NKCC2B, NKCC2A, and NKCC2F Bonferroni post test. P Ͻ 0.05 was considered significant. Transcripts by In Situ Hybridization For in situ hybridization, kidney sections were prepared as described Results previously (25). The mRNA expression of NKCC2A, -B, and -F was Mutated Transcripts in NKCC2BϪ/Ϫ Mice localized using digoxigenin-labeled riboprobes according to the man- NKCC2-deficient mice were viable and showed no gross ufacturer’s protocol (Roche). Sense and antisense probes were gener- anatomic, behavioral, or fertility abnormalities (Table 1). Cross- ated by in vitro transcription of a 350-bp cDNA of each isoform that ing of heterozygous mice yielded offspring of the different spanned from exon 4 into exon 5. In addition, a 96-bp antisense probe genotypes in near Mendelian ratio. In a first set of experiments, of each isoform from exon 4 was used. Sense probes were applied in we addressed the nature of the mutated NKCC2B transcripts parallel with antisense probes to test for unspecific hybridization sig- nals. and those of the remaining isoforms. As can bee seen in Figure 2, the targeting event led to the introduction of several prema- Immunohistochemistry ture stop codons in all reading frames, rendering NKCC2B Immunolabeling was performed as described (25) using guinea pig transcripts nonfunctional. NKCC2A and NKCC2F transcripts, anti-NKCC2 (directed against the C-terminal region; gift from Dr. in contrast, were found to be unaltered. Before removal of the Ellison, Oregon Health Sciences University, Portland, OR) followed by bulky neomycin-cassette, splicing of the mutated exon 4B was incubation with donkey anti-guinea pig cy3-Ig (Dianova, Hamburg, compromised, leading to two different transcripts with partial Germany). losses of neo (Figure 2A). In contrast, correct splicing was fully preserved after Cre-mediated removal of the floxed selection BP and Heart Rate cassette (Figure 2B). For assessment of whether targeting BP and heart rate in conscious mice were determined by tail-cuff NKCC2B would influence the quantity and the localization of manometry (Visitech Systems, Apex, NC) (26). the remaining NKCC2A and NKCC2F, their respective mRNA expression was determined by real-time RT-PCR of RNA that Blood Collection and Plasma Renin Determination was derived from the inner medulla, outer medulla, and cortex. Plasma renin concentration (PRC) in blood samples from tail veins As can been seen in Figure 3, targeting NKCC2B did not alter was measured with a commercial RIA kit (DiaSorin, Stillwater, MN), as the expression levels of the remaining NKCC2A and NKCC2F described previously (27). For assessment of renin secretion after inhi- isoforms. NKCC2F was the dominant isoform in the medulla bition of macula densa salt transport activity, a single dose of furo- with highest expression levels in the inner stripe of the outer semide (40 mg/kg) was injected intraperitoneally and blood samples medulla. NKCC2B was expressed mainly in the cortex and to were drawn 60 min later. For determination of macula densa–depen- dent inhibition of renin release, mice received a single intravenous (tail very low degrees in the outer medulla. NKCC2A was present in vein) injection of 1 ml of saline, and blood samples were collected 60 both cortex and medulla. NKCC2A mRNA levels in the outer min later (27). For chronic modulation of the renin- system, medulla were roughly twice those in the cortex. No NKCC2 mice were subjected to the following treatments: (1) Control group: mRNA was detected in the renal inner medulla. Mice were fed a standard rodent diet (0.3% NaCl [wt/wt]) for 1 wk; 2) salt-deficient group: After injection of a single dose of furosemide (40 Urine Osmolarity and Concentrating Ability mg/kg intraperitoneally), mice received a salt-deficient diet (0.03% To assess the effect of the loss of NKCC2B on urine-concen- NaCl [wt/wt]) for 1 wk; 3) and salt load group: Mice were fed a trating ability, we determined urine osmolarity both under high-salt diet (4% NaCl [wt/wt]) for 1 wk. ambient conditions and after 48 h of water deprivation (Figure 4). Ambient urine osmolarity averaged 1763 Ϯ 121 in wild-type Micropuncture Experiments mice and 1192 Ϯ 60 in NKCC2B-deficient mice (n ϭ 30 and 34, Measurements of stop flow pressure (PSF) during loop of Henle respectively; P Ͻ 0.0001). Water restriction substantially in- perfusion were done as described (12). When P had stabilized, loop of SF creased urine osmolarity in both wild-type (n ϭ 19) and Henle perfusion rate was increased to 30 nl/min, and maximum P SF NKCC2B knockout (n ϭ 20) mice to 3601 Ϯ 115 and 3311 Ϯ 97 responses were determined. Perfusion rates then were decreased to 20, P ϭ 15, 10, 5, and 0 nl/min and maintained until steady states were mmol/L, respectively ( 0.06). achieved at each flow rate. Logistic curve parameters were determined using GraphPad Prism (GraphPad, San Diego, CA). The perfusion fluid Plasma Renin and Plasma Aldosterone Concentrations To address the consequences of the loss of the macula densa contained 136 mM/L NaCl, 4 mM/L NaHCO3, 4 mM/L KCl, 2 mM/L

CaCl2, 7.5 mM/L urea, and 100 mg/100 ml FD&C green (Keystone, NKCC2B for the regulation of the renin system, we determined Bellefonte, PA). baseline PRC in NKCC2B-deficient mice, lacking NKCC2B in 2146 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 2143–2152, 2006

Table 1. Average concentrations of a number of plasma constituents in NKCC2Bϩ/ϩ and NKCC2BϪ/Ϫ mice (n ϭ 5 for each genotype)a

NKCC2Bϩ/ϩ NKCC2BϪ/Ϫ

Mean SEM Mean SEM

Glucose (mg/dl) 290.7 43.4 345.1 21.8 Cholesterol (mg/dl) 70.7 5.2 72.6 4.8 Triglycerides (mg/dl) 76.8 24.5 55.2 4.3 Na (mmol/L) 150.0 0.9 148.9 0.6 K (mmol/L) 4.4 0.1 4.1 0.1 Cl (mmol/L) 114.3 1.3 114.0 0.5 Ca (mmol/L) 2.2 0.03 2.2 0.03 Mg (mmol/L) 1.1 0.05 0.9 0.05 P (mg/dl) 7.9 0.5 7.7 0.5 Creatinine (mg/dl) 0.3 0.03 0.3 0.02 BUN (mg/dl) 21.8 1.4 17.8 1.6 Alkaline phosphatase (U/L) 59.0 8.3 41.1 2.0 ALT/GPT (U/L) 21.0 1.5 19.5 1.4 AST/GOT (U/L) 36.4 1.7 37.5 1.3 Creatinine kinase (U/L) 102.2 18.9 89.6 24.5 Uric acid (mg/dl) 2.0 0.3 1.5 0.3 Total protein (g/dl) 4.2 0.1 4.0 0.1

aALT/GPT, alanine aminotransferase/glutamate pyruvate transaminase; AST/GOT, aspartate aminotransferase/glutamate oxaloacetic transaminase; BUN, blood urea nitrogen. the macula densa and upstream parts of the TAL, and wild- in NKCC2BϪ/Ϫ mice (n ϭ 10). PRC was significantly lower type mice under control conditions and after acute inhibition of after saline injection in NKCC2BϪ/Ϫ compared with NKCC2 by administration of furosemide. Basal PRC levels NKCC2Bϩ/ϩ mice (P ϭ 0.0015). were similar in wild-type (n ϭ 30) and NKCC2BϪ/Ϫ mice (n ϭ Plasma aldosterone concentrations after exposure to high- or 22), averaging 1911Ϯ 150 and 1735 Ϯ 160 ng angiotensin I (Ang low-salt diet changed in parallel to PRC. As shown in Table 2, I)/ml per h (P ϭ 0.53). As can be seen in Figure 5, acute no significant differences between genotypes were observed administration of furosemide to inhibit all isoforms of NKCC2 under these conditions. increased PRC to the same extent in wild-type and knockout mice (7451 Ϯ 1016 [n ϭ 15] and 6213 Ϯ 449 ng Ang I/ml per h BP and Heart Rate [n ϭ 12], respectively; P ϭ 0.315 between genotypes). To inves- Systolic BP measured by the tail-cuff compression method in tigate the renin system under conditions of chronic modulation awake wild-type mice averaged 120 Ϯ 2.8 mmHg, and heart of oral salt intake, we determined PRC in mice that were fed rate was 610 Ϯ 19 bpm (n ϭ 10). In NKCC2B-deficient mice, either a high-salt diet or a salt-deficient diet for 1 wk (Figure 6). systolic BP and heart rate were 119 Ϯ 2.0 mmHg and 615 Ϯ 15 After the high-salt diet, PRC was slightly reduced in NKCC2B bpm, respectively (n ϭ 10; NS). wild-type mice from 2218 Ϯ 111 to 1832 Ϯ 174 ng Ang I/ml per h(n ϭ 12; P ϭ 0.038) versus control diet (Ϫ17%). In NKCC2B- Distal Tubular Chloride Concentration deficient mice, a high-salt diet lowered PRC more efficiently, To determine the effect of an NKCC2B deficiency on overall decreasing it from 2088 Ϯ 187 under normal diet to 1249 Ϯ 140 loop of Henle diluting ability, we determined chloride concentra- ng Ang I/ml per h (n ϭ 10; P ϭ 0.002 versus control diet and P ϭ tions in distal tubular fluid of superficial loops of Henle that were 0.019 versus wild-type), a reduction in PRC of 40%. Conversely, microperfused in situ at normal and elevated flow rates (Figure a low-salt diet stimulated PRC to similar levels in both 8A). At tubular perfusion rates of 6 or 15 nl/min, distal chloride NKCC2Bϩ/ϩ and Ϫ/Ϫ mice (3320 Ϯ 284 and 3359 Ϯ 511 ng concentrations in NKCC2BϪ/Ϫ exceeded those in wild-type mice Ang I/ml per h for wild-type [n ϭ 12] and knockout [n ϭ 10], by 22.0 and 13.1 mEq/L, averaging 88.8 Ϯ 4.3 versus 66.8 Ϯ 6.1 respectively; P ϭ 0.001 and 0.031 versus control diet). mmol/L at 6 nl/min and 106 Ϯ 6.2 versus 92.9 Ϯ 4.0 mEq/L at 15 For determination of the effect of an acute salt load on renin nl/min in NKCC2BϪ/Ϫ and wild-type mice, respectively (P ϭ secretion, mice received a single intravenous injection of 1 ml of 0.008 and P ϭ 0.042, between genotypes). Absolute Cl absorption saline, and blood samples were drawn 60 min later. As shown along the loop of Henle was significantly higher in wild-type than in Figure 7, acute salt loading suppressed PRC by 42%, from in NKCC2BϪ/Ϫ mice at the low flow rate (723 Ϯ 19 versus 602 Ϯ 1032 Ϯ 88 to 603 Ϯ 36 ng Ang I/ml per h, in wild-type (n ϭ 9), 26 pEq/min; P ϭ 0.001) but not at the high flow rate (1420 Ϯ 72 and by 64%, from 1139 Ϯ 149 to 408 Ϯ 52 ng Ang I/ml per h, versus 1269 Ϯ 100 pEq/min; P ϭ 0.22). Similarly, fractional Cl J Am Soc Nephrol 17: 2143–2152, 2006 NKCC2B Deletion and Kidney Function 2147

Figure 3. Quantification and localization of NKCC2B, NKCC2A, and NKCC2F mRNA in kidney regions of NKCC2Bϩ/ϩ and Ϫ/Ϫ mice; real-time PCR was performed using isoform-spe- cific TaqMan probes and 18s RNA as a reference. Data are given as percentage of the zone with highest expression levels of the respective isoforms. IM, inner medulla; OMIS, outer medulla inner stripe; OMOS, outer medulla outer stripe.

Figure 2. Sequencing of NKCC2B transcripts before and after re- moval of the neomycin resistance (neo) cassette. For both strains, the targeting event led to the introduction of several premature absorption was significantly higher in wild-type than in knockout stop codons in all reading frames (underlined italic base triplets), mice at the low but not at the high flow rate (Figure 8B). Water rendering NKCC2B transcripts nonfunctional. (A) Presence of the absorption along the loop of Henle also was significantly higher in neo cassette affected splicing of the mutated exon 4B and led to wild-type than knockout mice at the low flow rate (4.2 Ϯ 0.3 versus two different transcripts with partial losses of neo (* in the anno- 3.3 Ϯ 0.3 nl/min; P ϭ 0.032), whereas differences were NS at the tated sequence). (B) Correct splicing was observed after Cre-me- high flow rate (7.9 Ϯ 0.6 versus 7.5 Ϯ 0.7 nl/min). diated removal of the floxed selection cassette; gaps in the anno- tated sequence refer to remaining vector sequences. Arrows in the TGF Regulation of GFR schematic drawing of the transcripts (A and B) indicate PCR TGF responses were determined by measurement of proxi- primers used for cloning and sequencing. mal PSF during loop perfusion rates of 0, 5, 7.5, 10, 15, 20, and 2148 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 2143–2152, 2006

Figure 4. Ambient urine osmolarity and urine osmolarity after Figure 6. PRC in NKCC2Bϩ/ϩ and NKCC2BϪ/Ϫ mice that water deprivation in NKCC2Bϩ/ϩ and Ϫ/Ϫ mice. In water were fed a standard rodent diet (0.3% NaCl), high-salt diet (4% restriction experiments, female mice were used and urine was NaCl), or low-salt diet (a single dose of 40 mg/kg furosemide; collected by catheterization of the . 0.03% NaCl). PRC was determined after 1 wk of the respective treatment.

30 nl/min. Experiments were done in seven male NKCC2B wild-type and four male NKCC2B-deficient mice, with the 47.1 Ϯ 3.2, 46.1 Ϯ 2.6, 45.7 Ϯ 1.7, 41.5 Ϯ 1.9, and 38.8 Ϯ 2.1 number of examined being 13 and 11, respectively. mmHg in NKCC2BϪ/Ϫ mice (P ϭ 0.42, 0.14, 0.59, 0.04, 0.036, Body weight averaged 33 Ϯ 1.0 g in wild-type and 36 Ϯ 1.2gin and 0.07 for genotype comparison). Half-maximum TGF re- knockout mice (NS). Mean arterial BP during the period of sponses were calculated to be reached at a perfusion flow of micropuncture experiments was 95.1 Ϯ 2.4 and 94.1 Ϯ 3.0 12.3 Ϯ 0.6 in wild-type and of 15.2 Ϯ 1.2 nl/min in knockout mmHg for wild-type and knockout mice, respectively (NS). mice (P ϭ 0.033). Therefore, TGF response curves were right-

Mean PSF at zero perfusion was not significantly different be- shifted in NKCC2B-deficient mice (Figure 9). tween genotypes (43.1 Ϯ 2.1 versus 45.5 Ϯ 1.9 mmHg; P ϭ 0.42).

PSF at a saturating perfusion rate (30 nl/min) tended to be Localization of the NKCC2 Isoforms along the TAL lower in nephrons from wild-type compared with knockout In view of the largely preserved macula densa function in mice (31.9 Ϯ 2.3 and 37.1 Ϯ 2.6 mmHg; P ϭ 0.14). At interme- NKCC2B-deficient mice, we performed isoform-specific in situ diate perfusion rates of 5, 7.5, 10, 15, and 20 nl/min, PSF hybridizations to determine whether an additional isoform of decreased from 43.1 Ϯ 2.1 (zero perfusion) to 41.3 Ϯ 2.1, 43.6 Ϯ NKCC2 is present in macula densa cells. Using antisense ribo- 3.4, 39.5 Ϯ 2.0, 34.5 Ϯ 2.4, and 32.5 Ϯ 2.5 mmHg in wild-type probes that were specific for NKCC2B, NKCC2A, and NKCC2F mice, whereas it changed from 45.5 Ϯ 1.9 (zero perfusion) to mRNA, the macula densa segment of the TAL showed hybrid-

Figure 5. Plasma renin concentration (PRC) before and 60 min after acute administration of furosemide (40 mg/kg, intraperito- neally) in NKCC2Bϩ/ϩ (left) and Ϫ/Ϫ mice (right); lines connect data from the same mice. J Am Soc Nephrol 17: 2143–2152, 2006 NKCC2B Deletion and Kidney Function 2149

Figure 7. Effect of a single intravenous injection of 1 ml of saline on PRC in NKCC2Bϩ/ϩ (n ϭ 9) and Ϫ/Ϫ mice (n ϭ 10). Blood samples were taken 60 min after the injection. Lines connect values from the same animals; percentages indicate relative decrements of PRC.

Table 2. Plasma aldosterone concentrations (pg/ml) in NKCC2Bϩ/ϩ and Ϫ/Ϫ mice after 1 wk on control (0.3% NaCl), high-salt (4% NaCl), or low-salt diet (0.03% NaCl after a single dose of 40 mg/kg furosemide, intraperitoneally)

Normal n n n Aldosterone (pg/ml) Salt High Salt Low Salt

NKCC2Bϩ/ϩ 790 Ϯ 56 9 199 Ϯ 26 10 1327 Ϯ 130 9 NKCC2BϪ/Ϫ 672 Ϯ 43 10 159 Ϯ 23 10 1737 Ϯ 191 10

ization signals for both NKCC2B and NKCC2A but not experiments prove the feasibility of a specific inactivation of a NKCC2F (Figure 10). No signal was detected using respective splice variant of a gene by means of knock-in of premature stop sense probes (data not shown). Consistent with the presence of codons. We assume that this strategy can be used as a general the mRNA of an NKCC2 isoform other than NKCC2B, NKCC2 approach to obtain mouse models with inactivation of specific protein was detected in the macula densa of NKCC2B-deficient splice variants of proteins, although the generation of a nonfunc- mice (Figure 11). In line with our localization study on kidney tional protein may be more uncertain if the variable exon is segments using RT-PCR, NKCC2B and NKCC2A also were located close to the C-terminal end of the protein. detected in parts of the TAL upstream of the macula densa Whereas inactivation of the entire NKCC2 protein by gene segment (data not shown). targeting leads to a severe salt-losing nephropathy with most animals dying within the first 2 wk of postnatal life (22), specific Discussion inactivation of the B isoform of NKCC2 was not associated with Movement of NaCl mediated by NKCC2 is the major uptake gross structural or functional abnormalities. This is consonant mechanism in the TAL of the loop of Henle and macula densa with this and previous studies indicating that NKCC2B transcripts cells, and adequate NKCC2 operation is directly required for account for only approximately 10% of total NKCC2 mRNA ex- loop salt absorption and body Na balance, urine-concentrating pression (19). Ambient urine osmolarity and concentrating ability ability, renin secretion, and TGF. By alternative splicing, a after water restriction was only slightly compromised in number of different NKCC2 isoforms that show distinct spatial NKCC2B-deficient mice. Therefore, NKCC2B does not seem to distribution along the loop of Henle and that differ on their affinity for NaCl are generated (13–17). contribute importantly to the accumulation of medullary intersti- This report summarizes experiments in which we applied a tial solute concentrations that is the basis of the urine-concentrat- strategy to target NKCC2B without interfering with the expres- ing ability. In line with this finding are observations in a single sion of the remaining isoforms, NKCC2A and NKCC2F. Several Bartter patient with a missense mutation in exon 4B who dis- mouse models with specific removal of single exons have been played an unusually mild type I Bartter phenotype (33). Never- reported (23,29–32), but to our knowledge, this is the first report of theless, a significant increase in distal Cl concentration was ob- the inactivation of one of several isoforms derived from differen- served in distal tubular fluid of microperfused loops of Henle, tial splicing without affecting the mechanism of differential splic- suggesting an impairment of loop of Henle Cl absorption, pre- ing of the remaining isoforms. In our approach, we introduced sumably along the cortical TAL. It is of note that the effect of stop codons into the variable exon 4 (Figure 1), leading to prema- NKCC2B deficiency on diluting ability of individual loops of ture termination of translation of the B isoform of NKCC2. Our Henle was more pronounced at the low than at the high flow rate, 2150 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 2143–2152, 2006

Figure 8. (A) Chloride concentration in individual samples of distal tubular fluid of microperfused superficial loops of Henle in NKCC2Bϩ/ϩ (F) and NKCC2BϪ/Ϫ mice (E); data are shown for perfusion rates of 6 and 15 nl/min. (B) Mean fractional Cl absorption along the loop of Henle perfused at 6 and 15 nl/min. *P Ͻ 0.05. Figure 10. In situ hybridization of NKCC2 mRNA isoforms in the macula densa segment of the thick ascending limb (TAL; highlighted by arrows) in wild-type mice showing positive signals for NKCC2B and NKCC2A but not for the medullary F isoform of NKCC2.

cinity consistent with predominant expression of the B isoform in macula densa cells (13). NKCC2B has been suggested to represent the transporter isoform that primarily is responsible for detecting variations of tubular chloride concentration by the macula densa. We therefore examined whether specific NKCC2B deficiency affects TGF regulation of afferent arteriolar tone and renin secretion the two end points that are known to be altered by macula densa input. Our data show that maxi- Figure 9. Change of stop flow pressure (PSF) expressed as percent- mum TGF responses to increases in flow rate were not signif- age reduction from PSFmax (PSF at zero loop flow) in response to stepwise increases of loop of Henle flow rates in wild-type (F) and icantly different between NKCC2B-deficient and wild-type NKCC2BϪ/Ϫ mice (E). Flow rates that caused half-maximum mice. However, TGF responses in the mutant mice were re- Ͻ responses (V1/2) are indicated by vertical lines. *P 0.05. duced in the subnormal flow range, causing a right shift of the TGF curve. The requirement of NKCC2B to maintain a normal suggesting that NKCC2B may be more active in a low Cl concen- TGF responsiveness to subnormal flow changes indicates that tration range. NKCC2B seems to operate most efficiently in the range of low In situ hybridization in the mouse kidney has identified a NaCl concentrations. This observation is in general agreement punctate pattern of NKCC2B expression in the glomerular vi- with the recent demonstration that NKCC2B has a markedly J Am Soc Nephrol 17: 2143–2152, 2006 NKCC2B Deletion and Kidney Function 2151

ciency in the cortical TAL upstream of the macula densa seg- ment and salt loading is large enough to increase macula densa NaCl transport despite NKCC2B absence and that this inhibits renin release during conditions of salt loading. Micropuncture studies have shown that intravenous injections of isotonic sa- line indeed are accompanied with increased distal NaCl con- centrations (2,37). Overall, NKCC2B-deficient mice showed a mild phenotype compared with the massive salt-losing nephropathy observed in mice with inactivation of all NKCC2 isoforms (22). Further investigations will be necessary to distinguish clearly between a phenotype that is specifically related to loss of the B isoform of NKCC2 and a phenotype derived from the reduction of total NKCC2 expression as a result of NKCC2B deficiency.

Conclusion We generated mice that lack the B isoform of NKCC2 by introducing premature stop codons into exon 4B. NKCC2B- Figure 11. NKCC2 protein expression in the macula densa seg- deficient mice did not display a salt-losing phenotype, although ment of the TAL detected by an antibody directed against the ambient urine osmolarities were reduced. Distal Cl concentra- C-terminal end of the NKCC2 protein; staining was observed in both NKCC2Bϩ/ϩ (top) and NKCC2BϪ/Ϫ mice (bottom). tion was elevated and loop of Henle Cl absorption was reduced in microperfused superficial loops of Henle of NKCC2B-defi- cient mice. PRC in NKCC2B-deficient mice was reduced during higher Cl affinity than the other two isoforms (13,17). It remains salt loading. Maximum TGF responses were not significantly to be investigated whether a constant exposure of the macula reduced in NKCC2B-deficient mice, indicating the operation of densa cells to higher Cl concentrations in NKCC2B-deficient another isoform in the macula densa, presumably NKCC2A. mice, as suggested by our distal Cl concentration measure- However, NKCC2B absence caused a right shift of the TGF ments, leads to some desensitization of the macula densa cells, function curve consonant with dependence of TGF responsive- supporting a right shift of the TGF response curve. ness on NKCC2B in the low NaCl concentration range. Maintenance of maximum TGF responses in NKCC2B knock- out mice clearly indicates that NKCC2B is not the only isoform Acknowledgments present in macula densa cells. In fact, the present in situ hy- This work was supported by intramural funds from the National bridization evidence shows the coexpression of both NKCC2B Institute of Diabetes and Digestive and Kidney Diseases. M.O. is the and NKCC2A in the macula densa segment and upstream parts recipient of a Visiting Fellowship of the National Institute of Diabetes of the TAL. In addition, NKCC2 protein was found immuno- and Digestive and Kidney Diseases. H.C. was supported by a grant histochemically in the macula densa of NKCC2B-deficient mice from the Deutsche Forschungsgemeinschaft (SFB699/A4). with an antibody directed against the C-terminal part of the protein. Considering the different Cl affinities of the co-trans- Ϫ porter isoforms, with a K for Cl of 9 to 12 and 22 to 45 mM References m 1. Itoh S, Carretero OA: Role of the macula densa in renin for NKCC2B and NKCC2A, respectively (13,17,34), the A iso- release. Hypertension 7: I49–I54, 1985 form seems to be better suited to serve as a Cl sensor, with 2. Lorenz JN, Kotchen TA, Ott CE: Effect of Na and Cl infu- tubular Cl concentrations at the macula densa segment being sion on loop function and plasma renin activity in rats. approximately 35 mM (35). Am J Physiol 258: F1328–F1335, 1990 To explore the effect of NKCC2B deficiency on renin secre- 3. Lorenz JN, Weihprecht H, Schnermann J, Skott O, Briggs tion, the second end point of macula densa signaling, we de- JP: Renin release from isolated juxtaglomerular apparatus termined PRC in conscious knockout and wild-type mice. In- depends on macula densa chloride transport. Am J Physiol terference with NKCC2 function by loop diuretics is 260: F486–F493, 1991 consistently associated with a marked increase in renin secre- 4. Persson AE, Salomonsson M, Westerlund P, Greger R, tion, which is mediated in part by inhibition of macula densa Schlatter E, Gonzalez E: Macula densa cell function. Kidney salt transport capacity (36). Consistent with the notion that Int Suppl 32: S39–S44, 1991 5. Schnermann J, Briggs J: Concentration-dependent sodium macula densa function was largely intact in NKCC2B-deficient chloride transport as the signal in feedback control of glomer- mice as a result of the coexpression of NKCC2A, PRC values ular filtration rate. Kidney Int Suppl 12: S82–S89, 1982 were not significantly elevated in the knockout animals. In fact, 6. Skott O, Briggs JP: Direct demonstration of macula densa- PRC was found to be significantly reduced in NKCC2B-defi- mediated renin secretion. Science 237: 1618–1620, 1987 cient mice during both chronic and acute conditions of salt 7. Bell PD, Lapointe JY: Characteristics of membrane trans- loading. It is conceivable that the increase of macula densa port processes of macula densa cells. Clin Exp Pharmacol NaCl concentration caused by a combination of NKCC2B defi- Physiol 24: 541–547, 1997 2152 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 2143–2152, 2006

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