A mouse model of pseudohypoaldosteronism type II reveals a novel mechanism of renal tubular acidosis
Authors: Karen I. López-Cayuqueo, Maria Chavez-Canales, Alexia Pillot, Pascal Houillier, Maximilien Jayat, Jennifer Baraka-Vidot, Francesco Trepiccione, Véronique Baudrie, Cara Büsst, Christelle Soukaseum, Yusuke Kumai, Xavier Jeunemaître, Juliette Hadchouel, Dominique Eladari, Régine Chambrey
Journal: Kidney International (2018)

Pseudohypoaldosteronism type II (PHAII) is a genetic disease characterized by association of hyperkalemia, hyperchloremic metabolic acidosis, hypertension, low renin, and high sensitivity to thiazide diuretics. It is caused by mutations in the WNK1, WNK4, KLHL3 or CUL3 gene. There is strong evidence that excessive sodium chloride reabsorption by the sodium chloride cotransporter NCC in the distal convoluted tubule is involved. WNK4 is expressed not only in distal convoluted tubule cells but also in β−intercalated cells of the cortical collecting duct. These latter cells exchange intracellular bicarbonate for external chloride through pendrin, and therefore, account for renal base excretion. However, these cells can also mediate thiazide-sensitive sodium chloride absorption when the pendrin-dependent apical chloride influx is coupled to apical sodium influx by the sodium-driven chloride/bicarbonate exchanger. Here we determine whether this system is involved in the pathogenesis of PHAII. Renal pendrin activity was markedly increased in a mouse model carrying a WNK4 missense mutation (Q562E) previously identified in patients with PHAII. The upregulation of pendrin led to an increase in thiazide-sensitive sodium chloride absorption by the cortical collecting duct, and it caused metabolic acidosis. The function of apical potassium channels was altered in this model, and hyperkalemia was fully corrected by pendrin genetic ablation. Thus, we demonstrate an important contribution of pendrin in renal regulation of sodium chloride, potassium and acid-base homeostasis and in the pathophysiology of PHAII. Furthermore, we identify renal distal bicarbonate secretion as a novel mechanism of renal tubular acidosis. Pseudohypoaldosteronism type II (PHAII) is a genetic disease characterized by association of hyperkalemia, hyperchloremic metabolic acidosis, hypertension, low renin, and high sensitivity to thiazide diuretics. It is caused by mutations in the WNK1, WNK4, KLHL3 or CUL3 gene. There is strong evidence that excessive sodium chloride reabsorption by the sodium chloride cotransporter NCC in the distal convoluted tubule is involved. WNK4 is expressed not only in distal convoluted tubule cells but also in β−intercalated cells of the cortical collecting duct. These latter cells exchange intracellular bicarbonate for external chloride through pendrin, and therefore, account for renal base excretion. However, these cells can also mediate thiazide-sensitive sodium chloride absorption when the pendrin-dependent apical chloride influx is coupled to apical sodium influx by the sodium-driven chloride/bicarbonate exchanger. Here we determine whether this system is involved in the pathogenesis of PHAII. Renal pendrin activity was markedly increased in a mouse model carrying a WNK4 missense mutation (Q562E) previously identified in patients with PHAII. The upregulation of pendrin led to an increase in thiazide-sensitive sodium chloride absorption by the cortical collecting duct, and it caused metabolic acidosis. The function of apical potassium channels was altered in this model, and hyperkalemia was fully corrected by pendrin genetic ablation. Thus, we demonstrate an important contribution of pendrin in renal regulation of sodium chloride, potassium and acid-base homeostasis and in the pathophysiology of PHAII. Furthermore, we identify renal distal bicarbonate secretion as a novel mechanism of renal tubular acidosis. Pseudohypoaldosteronism type II (PHAII), also known as Gordon syndrome or familial hyperkalemic hypertension, is a rare genetic disease characterized by the association of hypertension, hyperchloremic metabolic acidosis, and hyperkalemia in the absence of renal failure.1Gordon R.D. Syndrome of hypertension and hyperkalemia with normal glomerular filtration rate.Hypertension. 1986; 8: 93-102Crossref PubMed Scopus (182) Google Scholar The pathogenesis of this syndrome has remained puzzling for decades because hyperkalemia along with renal metabolic acidosis are 2 features of hypoaldosteronism, whereas volume-dependent hypertension is observed in states caused by an excess of mineralocorticoids. A study by Schambelan et al. suggests that the disease is caused by excessive electroneutral absorption of chloride by the distal nephron.2Schambelan M. Sebastian A. Rector Jr., F.C. Mineralocorticoid-resistant renal hyperkalemia without salt wasting (type II pseudohypoaldosteronism): role of increased renal chloride reabsorption.Kidney Int. 1981; 19: 716-727Abstract Full Text PDF PubMed Scopus (193) Google Scholar The observation that most symptoms of PHAII can be corrected by thiazide diuretics supports the possibility that a gain of function of the thiazide-sensitive sodium chloride (NaCl) cotransporter NCC of the distal convoluted tubule (DCT) plays a central role in PHAII. A breakthrough in our understanding of this disease came in 2001 from the discovery that mutations in genes encoding 2 serine-threonine kinases of the WNK family, WNK1 and WNK4, account for PHAII in some patients.3Wilson F.H. Disse-Nicodeme S. Choate K.A. et al.Human hypertension caused by mutations in WNK kinases.Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1217) Google Scholar WNK1 and WNK4 are elements of a complex signaling network involved in the regulation of ion transport in the distal nephron.4Hadchouel J. Ellison D.H. Gamba G. Regulation of renal electrolyte transport by WNK and SPAK-OSR1 kinases.Annu Rev Physiol. 2016; 78: 367-389Crossref PubMed Scopus (139) Google Scholar PHAII mutations in the WNK1 gene are large intronic deletions that cause an increase in WNK1 expression, whereas WNK4 mutations are missense mutations clustered in a short acidic segment distal to the kinase domain.3Wilson F.H. Disse-Nicodeme S. Choate K.A. et al.Human hypertension caused by mutations in WNK kinases.Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1217) Google Scholar These missense mutations also result in increased expression of WNK4 due to a decrease in degradation of the protein.5Kahle K.T. Wilson F.H. Leng Q. et al.WNK4 regulates the balance between renal NaCl reabsorption and K+ secretion.Nat Genet. 2003; 35: 372-376Crossref PubMed Scopus (342) Google Scholar The ubiquitin-ligase complex formed by the proteins KLHL3 and CUL3 has been identified as responsible for this degradation,4Hadchouel J. Ellison D.H. Gamba G. Regulation of renal electrolyte transport by WNK and SPAK-OSR1 kinases.Annu Rev Physiol. 2016; 78: 367-389Crossref PubMed Scopus (139) Google Scholar and mutations in both proteins have been recently identified in patients with PHAII.6Boyden L.M. Choi M. Choate K.A. et al.Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities.Nature. 2012; 482: 98-102Crossref PubMed Scopus (459) Google Scholar, 7Louis-Dit-Picard H. Barc J. Trujillano D. et al.KLHL3 mutations cause familial hyperkalemic hypertension by impairing ion transport in the distal nephron.Nat Genet. 2012; 44 (S451–S453): 456-460Crossref PubMed Scopus (252) Google Scholar Logically, the studies conducted over the past 15 years have focused on the regulation of NCC by WNK1 and WNK4. Transgenic mice expressing a WNK4 transgene carrying 1 of the missense mutations identified in PHAII patients (WNK4-Q562E; TgWnk4PHAII mice) displayed all clinical features of the syndrome.8Lalioti M.D. Zhang J. Volkman H.M. et al.Wnk4 controls blood pressure and potassium homeostasis via regulation of mass and activity of the distal convoluted tubule.Nat Genet. 2006; 38: 1124-1132Crossref PubMed Scopus (309) Google Scholar The same phenotypes were seen in a Wnk4(D561A/+) knock-in mouse model9Yang S.S. Morimoto T. Rai T. et al.Molecular pathogenesis of pseudohypoaldosteronism type II: generation and analysis of a Wnk4(D561A/+) knockin mouse model.Cell Metab. 2007; 5: 331-344Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar and in Wnk1+/FHHt mice, another PHAII model in which the first intron of Wnk1 is deleted.10Vidal-Petiot E. Elvira-Matelot E. Mutig K. et al.WNK1-related Familial Hyperkalemic Hypertension results from an increased expression of L-WNK1 specifically in the distal nephron.Proc Natl Acad Sci U S A. 2013; 110: 14366-14371Crossref PubMed Scopus (91) Google Scholar In these models, increased NCC phosphorylation and expression at the apical cell membrane result in enhanced NaCl absorption by the DCT, and vascular volume expansion, favoring the onset of hypertension. Conversely, Wnk4 knock-out mice exhibited a mild Gitelman-like syndrome, with normal blood pressure, increased plasma renin activity, and reduced NCC expression and phosphorylation.11Castaneda-Bueno M. Cervantes-Perez L.G. Vazquez N. et al.Activation of the renal Na+:Cl- cotransporter by angiotensin II is a WNK4-dependent process.Proc Natl Acad Sci U S A. 2012; 109: 7929-7934Crossref PubMed Scopus (194) Google Scholar The aforementioned studies confirm the importance of NCC in the pathogenesis of PHAII syndrome. However, other studies have suggested that increased NCC activity or abundance is not sufficient to cause PHAII. In transgenic mice overexpressing NCC, NCC abundance was increased but the mice displayed a phenotype similar to wild-type mice.12McCormick J.A. Nelson J.H. Yang C.L. et al.Overexpression of the sodium chloride cotransporter is not sufficient to cause familial hyperkalemic hypertension.Hypertension. 2011; 58: 888-894Crossref PubMed Scopus (37) Google Scholar Additionally, mice inactivated for KS-WNK1 or Nedd4-2 did not exhibit hyperkalemic hypertension despite increased NCC expression and phosphorylation.13Hadchouel J. Soukaseum C. Busst C. et al.Decreased ENaC expression compensates the increased NCC activity following inactivation of the kidney-specific isoform of WNK1 and prevents hypertension.Proc Natl Acad Sci U S A. 2010; 107: 18109-18114Crossref PubMed Scopus (81) Google Scholar, 14Ronzaud C. Loffing-Cueni D. Hausel P. et al.Renal tubular NEDD4-2 deficiency causes NCC-mediated salt-dependent hypertension.J Clin Invest. 2013; 123: 657-665PubMed Google Scholar In the kidney, WNK4 is expressed not only in the DCT, but has also been found in the cortical collecting duct (CCD).3Wilson F.H. Disse-Nicodeme S. Choate K.A. et al.Human hypertension caused by mutations in WNK kinases.Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1217) Google Scholar, 15Ohno M. Uchida K. Ohashi T. et al.Immunolocalization of WNK4 in mouse kidney.Histochem Cell Biol. 2011; 136: 25-35Crossref PubMed Scopus (40) Google Scholar, 16Shibata S. Rinehart J. Zhang J. et al.Mineralocorticoid receptor phosphorylation regulates ligand binding and renal response to volume depletion and hyperkalemia.Cell Metab. 2013; 18: 660-671Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Alongside the amiloride-sensitive epithelial sodium channel ENaC, which is expressed by principal cells, we have demonstrated the presence of a novel thiazide-sensitive electroneutral NaCl uptake mechanism in renal intercalated cells (ICs) of the CCD.17Leviel F. Hubner C.A. Houillier P. et al.The Na+-dependent chloride-bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ reabsorption process in the renal cortical collecting ducts of mice.J Clin Invest. 2010; 120: 1627-1635Crossref PubMed Scopus (244) Google Scholar NaCl is taken up from urine by the coordinated action of the Cl–/HCO3– exchanger pendrin/SLC26A4 and the sodium ion (Na+)-driven Cl–/2HCO3– exchanger NDCBE/SLC4A8. Overexpression of pendrin in ICs can favor the onset of chloride-dependent hypertension, indicating that this system likely plays a role in the regulation of blood pressure in vivo.18Jacques T. Picard N. Miller R.L. et al.Overexpression of pendrin in intercalated cells produces chloride-sensitive hypertension.J Am Soc Nephrol. 2013; 24: 1104-1113Crossref PubMed Scopus (72) Google Scholar Conversely, pendrin-deficient mice are protected against mineralocorticoid-induced hypertension19Verlander J.W. Hassell K.A. Royaux I.E. et al.Deoxycorticosterone upregulates PDS (Slc26a4) in mouse kidney: role of pendrin in mineralocorticoid-induced hypertension.Hypertension. 2003; 42: 356-362Crossref PubMed Scopus (206) Google Scholar and develop a lower blood pressure during NaCl restriction.20Wall S.M. Kim Y.H. Stanley L. et al.NaCl restriction upregulates renal Slc26a4 through subcellular redistribution: role in Cl- conservation.Hypertension. 2004; 44: 982-987Crossref PubMed Scopus (166) Google Scholar Importantly, acute inactivation of pendrin results in a lower blood pressure.21Trepiccione F. Soukaseum C. Baudrie V. et al.Acute genetic ablation of pendrin lowers blood pressure in mice.Nephrol Dial Transplant. 2017; 32: 1137-1145PubMed Google Scholar Finally, we demonstrated that NCC compensated for NDCBE inactivation and that double deletion of these proteins in mouse induces hypokalemia.22Sinning A. Radionov N. Trepiccione F. et al.Double knockout of the Na+-driven Cl-/HCO3- exchanger and Na+/Cl- cotransporter induces hypokalemia and volume depletion.J Am Soc Nephrol. 2017; 28: 130-139Crossref PubMed Scopus (37) Google Scholar Therefore, we hypothesized that this novel system might be involved in the pathogenesis of PHAII. In the present study, we demonstrate that PHAII-causing mutation of WNK4 increases pendrin activity and pendrin/NDCBE-dependent NaCl absorption. We also demonstrate that pendrin hyperactivity is necessary to drive hyperchloremic metabolic acidosis and hyperkalemia in PHAII because both symptoms are corrected by pendrin genetic ablation. As shown in Figure 1a–e , TgWnk4PHAII mice exhibit the phenotypic abnormalities characteristic of PHAII: metabolic hyperchloremic acidosis and hyperkalemia. Urinary aldosterone excretion was significantly increased in TgWnk4PHAII mice (Figure 1f), whereas renin mRNA abundance was lower in the PHAII mice (Figure 1g) as observed in most patients experiencing PHAII. As shown by others,8Lalioti M.D. Zhang J. Volkman H.M. et al.Wnk4 controls blood pressure and potassium homeostasis via regulation of mass and activity of the distal convoluted tubule.Nat Genet. 2006; 38: 1124-1132Crossref PubMed Scopus (309) Google Scholar, 9Yang S.S. Morimoto T. Rai T. et al.Molecular pathogenesis of pseudohypoaldosteronism type II: generation and analysis of a Wnk4(D561A/+) knockin mouse model.Cell Metab. 2007; 5: 331-344Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar NCC and phosphor-T53 NCC protein abundance was dramatically increased in TgWnk4PHAII mice (Figure 1h). Transepithelial fluxes of Na+ (JNa), potassium ion (K+; JK), chloride ion (Cl–; JCl) and the transepithelial voltage (Vte) were measured in microperfused CCDs isolated from TgWnk4PHAII mice fed a standard diet (Figure 2). No transport activity was detectable in CCDs isolated from control mice, as previously described.17Leviel F. Hubner C.A. Houillier P. et al.The Na+-dependent chloride-bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ reabsorption process in the renal cortical collecting ducts of mice.J Clin Invest. 2010; 120: 1627-1635Crossref PubMed Scopus (244) Google Scholar However, CCDs isolated from TgWnk4PHAII mice exhibited net NaCl absorption but did not develop a significant lumen negative Vte and did not secrete K+, suggesting the absence of detectable ENaC activity. Furthermore, NaCl absorption was fully inhibited by luminal addition of 100 μM hydrochlorothiazide (HCTZ). Taken together, these experiments demonstrate that electroneutral NaCl transport mediated by pendrin/NDCBE is stimulated by PHAII-causing mutation of WNK4. ENaC activity is generally detectable not in the CCD but only in the connecting tubule (CNT), a nephron segment, which is not suitable for in vitro microperfusion. Thus, to estimate the total ENaC activity, we next measured the effect of an acute amiloride injection in TgWnk4PHAII and control mice on urinary Na+ and K+ excretion. The natriuretic response to amiloride was identical for both groups (Figure 3a, left panel), suggesting that Na+ reabsorption through ENaC is not altered in TgWnk4PHAII mice. However, amiloride had significantly less effect on urinary K+ excretion in TgWnk4PHAII mice than in controls (Figure 3a, right panel). These experiments demonstrate that ENaC-dependent K+ transport mechanisms per se are inhibited in TgWnk4PHAII mice. We next examined the relative protein abundance of the α and γ subunits of ENaC in TgWnk4PHAII and control mice by immunoblot analyses of plasma membrane-enriched preparations isolated from renal cortex (Figure 3b). The abundance of the full-length 90 kDa form and the cleaved N-terminal 30 kDa fragment of α-ENaC was higher in TgWnk4PHAII mice. The abundance of the full-length form of γ-ENaC (85 kDa) was decreased in TgWnk4PHAII mice, whereas the abundance of the cleaved 70 kDa form of γ-ENaC was increased. These alterations are indicative of an activation of ENaC possibly by aldosterone. Hyperkalemia can cause renal tubular acidosis.23DuBose Jr., T.D. Hyperkalemic hyperchloremic metabolic acidosis: pathophysiologic insights.Kidney Int. 1997; 51: 591-602Abstract Full Text PDF PubMed Scopus (54) Google Scholar To check the importance of high plasma potassium in the development of acidosis in PHAII, we tested whether normalization of plasma potassium concentration can alleviate the acidosis of TgWnk4PHAII mice. TgWnk4PHAII mice were fed with a synthetic low-K+ diet for 4 days (Figure 4). As expected, plasma K+ was normalized following this treatment. However, the decrease in plasma K+ did not modify either plasma HCO3– or pH, indicating that acidosis in PHAII is not caused primarily by hyperkalemia. We next evaluated whether metabolic acidosis in TgWnk4PHAII mice is due to impaired proton secretion by the distal nephron. Acid was administrated as NH4Cl 0.28 M in the drinking water for 15 days. Figure 5 shows that before acid loading, only plasma HCO3– (panel b) was significantly lower in TgWnk4PHAII mice, whereas urine pH (panel c), urinary titrable acid excretion (panel d), and ammonium excretion (panel e) were identical in TgWnk4PHAII and control mice. After 2 days of acid loading, blood pH and HCO3– decreased in both groups. However, TgWnk4PHAII mice developed a much stronger metabolic acidosis. When acid loading was continued, control mice were able to cope with the acid load and exhibited normal blood pH and HCO3– at day 15 of the treatment. By contrast, severe metabolic acidosis persisted in TgWnk4PHAII mice. Interestingly, following acid loading, urine pH decreased and ammonium excretion increased maximally in both groups, indicating that proton secretion, and ammonium transport or metabolism, are normal in these mice. The effect of acid loading was also tested in WNK1+/FHHt mice, another PHAII model in which the first intron of WNK1 is deleted.10Vidal-Petiot E. Elvira-Matelot E. Mutig K. et al.WNK1-related Familial Hyperkalemic Hypertension results from an increased expression of L-WNK1 specifically in the distal nephron.Proc Natl Acad Sci U S A. 2013; 110: 14366-14371Crossref PubMed Scopus (91) Google Scholar When submitted to an acid load (Supplementary Figure S1), these mice responded exactly like TgWnk4PHAII mice, indicating that the same mechanism is causing acidosis in both PHAII models independently of the mutation. Finally, Figure 5f shows a marked shift in the relationship between urinary ammonium excretion and blood bicarbonate concentration (reflecting the severity of metabolic acidosis) in TgWnk4PHAII versus control mice. This demonstrated that, while proton secretion or ammonium excretion capabilities are preserved in TgWnk4PHAII mice, these mice clearly have a defect in renal net acid excretion. This led us to hypothesize that metabolic acidosis in PHAII is not caused by impaired acid excretion but rather is due to increased renal loss of base. We next examined pendrin activity by measuring Cl–-dependent alkalinization in ICs in CCDs isolated from TgWnk4PHAII and control mice (Figure 5g, left panel). The rate of intracellular alkalinization in response to luminal Cl– removal, an estimate of apical Cl–/HCO3– exchange activity, was much higher in ICs from TgWnk4PHAII mice than in control mice (Figure 5g, right panel). These results indicate that pendrin activity per cell is increased in this model. Further, immunofluorescence staining revealed an increase in pendrin labeling in the collecting ducts located in renal medullary rays of TgWnk4PHAII mice (Figure 5h). ICs exist in 3 different forms: (i) α-ICs characterized by apical expression of the V-ATPase and basolateral expression of an anion exchanger, which is an alternately spliced product of the erythrocyte AE1 gene, (ii) β-ICs, which harbor apical pendrin and basolateral V-ATPase, and (iii) non-α-, non-β-ICs with apical pendrin and apical V-ATPase. The total number of ICs remains generally constant; however, the relative number of α-ICs versus β-ICs is tightly controlled and adapted depending on the acid-base status.24Schwartz G.J. Barasch J. Al-Awqati Q. Plasticity of functional epithelial polarity.Nature. 1985; 318: 368-371Crossref PubMed Scopus (260) Google Scholar We therefore evaluated the number of the different versions of ICs in the CCDs of control and TgWnk4PHAII mice using triple immunofluorescence labeling for pendrin, for the E subunit of the V-ATPase, and for AE1 (Figure 5h). In both transgenic and control mice, CCDs located in medullary rays exhibited α- and β-ICs but no non-α-, non-β-ICs. We counted 1867 ICs (defined as those that stained for the E subunit of the V-ATPase) from 4 independent control mice, and the results are displayed as white bars in Figure 5i. Of these ICs, 37% stained for pendrin, whereas 63% had AE1 staining. In 4 independent TgWnk4PHAII mice, we counted 1955 ICs (black bars in Figure 5i). The fraction of β-ICs was significantly increased in CCDs of TgWnk4PHAII mice as 50% of ICs were pendrin-positive. Conversely, the number of α-ICs was reduced and the total number of ICs was not different between control and TgWnk4PHAII mice. Because pendrin activity per cell is increased, we can conclude that total pendrin activity is increased in CCDs of mutant mice. As shown in Figure 5j, this increased pendrin transport activity was associated with a significant increase in CCD fractional volume. To verify whether hyperactivity of pendrin participates to PHAII phenotype, we next tested the effects of pendrin disruption in mice harboring the mutation of WNK4 causing PHAII. Double-mutant mice (TgWnk4PHAII;Pds–/–) no longer exhibited hyperchloremic metabolic acidosis and hyperkalemia (Table 1). These results confirmed that activation of pendrin participates to hyperchloremic acidosis and hyperkalemia in PHAII.Table 1Blood parameters of control, TgWnk4PHAII;Pds+/+, and TgWnk4PHAII;Pds–/– miceBloodControlTgWnk4PHA2;Pds+/+TgWnk4PHA2;Pds–/–pH7.26 ± 0.017.26 ± 0.017.30 ± 0.01PCO256 ± 151 ± 1∗54 ± 2HCO3–, mM22.9 ± 0.720.8 ± 0.3∗23.7 ± 0.6##Na+, mM148.5 ± 1.0148.6 ± 0.6149.9 ± 0.4Cl–, mM109.1 ± 0.4114.0 ± 0.4∗∗112.0 ± 0.5K+, mM4.40 ± 0.075.08 ± 0.08∗∗4.39 ± 0.06##Hematocrit, %42.5 ± 0.839.8 ± 0.441.8 ± 0.5Cl–, chloride ion; HCO3–, bicarbonate ion; K+, potassium ion; Na+, sodium ion; PCO2, partial pressure of carbon dioxide.Values are means ± SEM; N = 16, 39, and 29 for control, TgWnk4;Pds+/+, and TgWnk4;Pds-/- mice, respectively. Statistical significance between groups was assessed by analysis of variance following Bonferroni's multiple comparison test when appropriate. Asterisks indicate statistical significance versus control mice (*P < 0.05, **P < 0.01, and ****P < 0.0001). The # symbols indicate statistical significance versus TgWnk4;Pds+/+ mice (##P < 0.01 and ####P < 0.0001). No significant difference was observed between control and TgWnk4;Pds-/- mice. Open table in a new tab Cl–, chloride ion; HCO3–, bicarbonate ion; K+, potassium ion; Na+, sodium ion; PCO2, partial pressure of carbon dioxide. Values are means ± SEM; N = 16, 39, and 29 for control, TgWnk4;Pds+/+, and TgWnk4;Pds-/- mice, respectively. Statistical significance between groups was assessed by analysis of variance following Bonferroni's multiple comparison test when appropriate. Asterisks indicate statistical significance versus control mice (*P < 0.05, **P < 0.01, and ****P < 0.0001). The # symbols indicate statistical significance versus TgWnk4;Pds+/+ mice (##P < 0.01 and ####P < 0.0001). No significant difference was observed between control and TgWnk4;Pds-/- mice. Pendrin disruption in TgWnk4PHAII mice led to a significant increase in hematocrit (Table 1). Renin mRNA was also higher in double transgenic TgWnk4PHAII;Pds–/– mice (Figure 6). No significant difference, however, was detected in blood pressure measured by telemetry (Figure 6) or in aldosterone levels (7.26 ± 0.57 nM/mM creatinine in TgWnk4PHAII;Pds–/– mice, n = 10 vs. 7.93 ± 0.63 nM/mM creatinine in TgWnk4PHAII;Pds+/+ mice, n = 12, NS). The effects of pendrin disruption on blood pressure remained limited, presumably because NCC is still markedly stimulated. Accordingly, no change in protein abundance of NCC and phosphor-T53 NCC was observed between TgWnk4PHAII;Pds+/+ and TgWnk4PHAII;Pds–/– mice by immunoblot analyses of plasma membrane-enriched preparations isolated from renal cortex (Figure 7a). Protein abundance of ENaC subunits was also assessed. Both α and γ subunits decreased in TgWnk4PHAII;Pds–/– mice compared with TgWnk4PHAII;Pds+/+ mice, consistent with the effect of isolated deletion of pendrin on ENaC expression described previously.25Kim Y.H. Pech V. Spencer K.B. et al.Reduced ENaC protein abundance contributes to the lower blood pressure observed in pendrin-null mice.Am J Physiol Renal Physiol. 2007; 293: F1314-F1324Crossref PubMed Scopus (85) Google ScholarFigure 7Effect of pendrin deletion in TgWnk4PHAII mice on sodium ion (Na+) and chloride ion (Cl-) transporters. (a) Immunoblots performed on plasma membrane-enriched fraction of renal cortex from TgWnk4;Pds+/+ (N = 6) and TgWnk4;Pds-/- (N = 6) mice. Bar graph shows a summary of densitometric analyses. Densitometric values were normalized to the mean for the TgWnk4;Pds+/+ mice that was defined as 100%, and results were expressed as mean ± SEM. Statistical significance was assessed by unpaired Student t-test. (b) Effect of amiloride injection on urinary Na+ and potassium ion (K+) excretion in TgWnk4;Pds+/+ (N = 7) and TgWnk4;Pds-/- (N = 5) mice. Amiloride elicits significant increase in Na+ excretion and decrease in K+ excretion in both TgWnk4;Pds+/+ and TgWnk4;Pds-/- mice 6 hours after injection. Amiloride induced natriuresis was not different between groups. K+ excretion after amiloride was significantly lower in TgWnk4;Pds-/- mice, indicating that epithelial sodium channel (ENaC)-dependent K+ secretion is increased in these mice. Values are the mean ± SEM. Statistical significance was assessed by 1-way analysis of variance. **P < 0.01 and ****P < 0.0001, vehicle versus amiloride. ****P < 0.0001, TgWnk4;Pds+/+ versus TgWnk4;Pds-/- mice following amiloride injection. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next measured total ENaC-dependent Na+ absorption and K+ secretion in double-mutant mice by assessing the effects of an acute injection of amiloride on urinary excretion of Na+ and K+. The natriuretic response to amiloride (Figure 7b, left panel) was not different between TgWnk4PHAII;Pds+/+ and TgWnk4PHAII;Pds–/– mice, indicating that ENaC activity is not altered by pendrin disruption. However, the decrease in urinary K+ excretion following amiloride was higher in TgWnk4PHAII;Pds–/– mice than in TgWnk4PHAII;Pds+/+ mice (Figure 7b, right panel), suggesting that ENaC-dependent K+ secretion is increased after pendrin disruption in TgWnk4PHAII mice. Studies performed either in humans or in animal models have confirmed that PHAII-causing mutations lead to increased phosphorylation, membrane expression, and activity of the cotransporter NCC of the DCT cells. β-ICs exchange HCO3– for Cl– through the apical Cl–/HCO3– exchanger pendrin/SLC26A4, fulfilling their primary function of HCO3– excretion. When pendrin is functionally coupled with the Na+-driven Cl–/2HCO3– exchanger NDCBE/SLC4A8, thiazide-sensitive, electroneutral NaCl absorption then occurs in these cells.17Leviel F. Hubner C.A. Houillier P. et al.The Na+-dependent chloride-bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ reabsorption process in the renal cortical collecting ducts of mice.J Clin Invest. 2010; 120: 1627-1635Crossref PubMed Scopus (244) Google Scholar We therefore performed this study to determine to what extent pendrin might participate to the PHAII phenotype. Here we show that pendrin activity and thiazide-sensitive electroneutral NaCl absorption through the pendrin/NDCBE transport system are activated in isolated CCDs of TgWnk4PHAII mice. Elucidating the mechanism of pendrin activation was beyond the scope of this study. However, several studies in the literature support the idea that WNK4 is involved in pendrin activation. For instance, the pendrin/NDCBE transport system is the dominant mechanism accounting for Na+ absorption in the CCD of Ncc knockout mice,17Leviel F. Hubner C.A. Houillier P. et al.The Na+-dependent chloride-bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ reabsorption process in the renal cortical collecting ducts of mice.J Clin Invest. 2010; 120: 1627-1635Crossref PubMed Scopus (244) Google Scholar whereas in Wnk4 knockout mice, which exhibit dramatically low NCC expression,