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Tacrolimus ameliorates the phenotypes of type 4 Bartter syndrome model mice through activation of sodium–potassium–2 chloride cotransporter and sodium–chloride cotransporter
Biochemical and Biophysical Research Communications 517 (2019) 364e368
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Tacrolimus ameliorates the phenotypes of type 4 Bartter syndrome model mice through activation of sodiumepotassiume2 chloride cotransporter and sodiumechloride cotransporter Yoshiaki Matsuura, Naohiro Nomura*, Wakana Shoda, Takayasu Mori, Kiyoshi Isobe, Koichiro Susa, Fumiaki Ando, Eisei Sohara, Tatemitsu Rai, Shinichi Uchida Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo, 1138519, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 July 2019 Accepted 22 July 2019 Available online 27 July 2019
Type 4 Bartter syndrome (BS) is caused by genetic mutations in barttin, which is coded for by BSND. Barttin serves as the b-subunit of the ClCeK chloride (Cl) channel, which is widely expressed in distal nephrons. Type 4 BS is characterized by severely impaired reabsorption of salt, which may cause polyuria, hypokalemia, and metabolic alkalosis. Calcineurin inhibitors reportedly induce renal salt retention and hyperkalemia by enhancing the phosphorylation of the sodium (Naþ)epotassium (Kþ)e2Cl cotransporter (NKCC2) and NaþeCl cotransporter (NCC). In addition, we have previously reported that tacrolimus, a calcineurin inhibitor, increases the levels of phosphorylated NCC. In this study, we administered tacrolimus to barttin hypomorphic (Bsndneo/neo) mice, a murine model of type 4 BS that exhibits polyuria, hypokalemia, and metabolic alkalosis. Administration of tacrolimus increased the serum Kþ level and suppressed urinary Kþ excretion. Furthermore, after treatment with tacrolimus, Bsndneo/neo mice increased levels of phosphorylated NCC and NKCC2. We conclude that tacrolimus partially improves clinical phenotypes of Bsndneo/neo mice, and that calcineurin inhibitors might be effective for treating type 4 BS. © 2019 Elsevier Inc. All rights reserved.
Keywords: Bartter syndrome Calcineurin inhibitor Hypokalemia Sodiumechloride cotransporter
1. Introduction Type 4 Bartter syndrome (BS) is characterized by very severe symptoms, including fetal polyuria, which causes maternal polyhydramnios; postnatal polyuria, which causes dehydration; hypokalemic metabolic alkalosis, and sensorineural deafness. Type 4 BS is caused by mutation of barttin (coded by BSND), which is the bsubunit of the ClCeKa/1 and ClCeKb/2 channels [1,2]. Because barttin is widely expressed from the thin ascending loop of Henle to the collecting duct, mutations in BSND lead to severe impairments in renal function. The thick ascending loop of Henle (TAL) and the distal convoluted tubule (DCT) are important for regulating the urinary excretion of electrolytes in the nephron. In the TAL and DCT, sodium (Naþ) and chloride (Cl) are reabsorbed through the Naþepotassium (Kþ)e2Cl cotransporter (NKCC2) and NaþeCl cotransporter (NCC) in conjunction with ClCeKb/2 and barttin. One
* Corresponding author. E-mail address: [email protected] (N. Nomura). https://doi.org/10.1016/j.bbrc.2019.07.086 0006-291X/© 2019 Elsevier Inc. All rights reserved.
commonly used treatment for type 4 BS is the administration of Kþ supplements for hypokalemia. In addition to Kþ supplementation, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARB), and Kþ-sparing diuretics are also used to suppress urinary Kþ excretion. Nonsteroidal anti-inflammatory drugs (NSAIDs) are also used to suppress levels of prostaglandins and decrease urinary volume and Naþ excretion in refractory cases of type 4 BS [3,4]. However, there are still problems associated with these treatments. For instance, Kþ supplements are often needed in high doses, ACE inhibitors and ARB are not tolerated in patients hypotension, and NSAIDs may cause gastrointestinal and kidney damage. Although activators of type 4 BS-related transporters, such as NKCC2 or NCC, could be promising drugs, there are currently no commercially available activators of NKCC2 nor NCC. Inhibitors of calcineurin, such as tacrolimus and cyclosporine, are often used as immunosuppressants to prevent rejection against transplanted organs. Previous studies have shown that calcineurin inhibitors may induce hyperkalemia and hypertension as sideeffects [5]. Studies showing that calcineurin inhibitors may affect
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levels of NCC phosphorylation and salt-sensitive hypertension have also been reported. Hoorn et al. have reported that treatment with tacrolimus, a calcineurin inhibitor, significantly increased NCC phosphorylation in the murine and human kidney, thus leading to salt-sensitive hypertension [6]. Tacrolimus inhibits calcineurin by binding to the 12 kDa FK506-binding protein (FKBP12). Mice lacking FKBP12 in the nephron did not show tacrolimus-induced hypertension or increased levels of phosphorylated NCC [7]. We have previously reported that tacrolimus inhibited high- Kþ-induced NCC dephosphorylation during the acute phase of Kþ load [8]. Calcineurin inhibitors have been shown to increase the phosphorylation of NKCC2 in some rodent studies [9e11]. This study tested the hypothesis that tacrolimus would improve the clinical phenotypes of a type 4 BS mouse model by increasing the phosphorylation of NKCC2 and NCC. We used Bsndneo(R8L)/neo(R8L) (Bsndneo/neo) mice as a type 4 BS model, which have a hypomorphic mutation in BSND that causes symptoms of BS such as polyuria, hypokalemia and metabolic alkalosis [12]. 2. Materials and methods 2.1. Animal experiences All experiments were performed according to the Tokyo Medical and Dental University Animal Experiment Guidelines. The protocol was approved by The Animal Care and Use Committee of Tokyo Medical and Dental University. Bsndneo/neo mice (C57BL6 background) were used as a loss-of-function model of type 4 BS. We used mice of both sexes between the ages of 8e16 weeks, and all animals weighed between 16 and 28 g. These parameters were matched in each group. All mice were fed according to the CL-2 diet (Oriental Yeast Co., Tokyo, Japan). Tacrolimus (Toronto Research Chemicals Inc., Toronto, Canada) was dissolved in 75% dimethyl sulfoxide (DMSO) and administered once a day for a period of one week (2 mg/kg/day s.c.). The equivalent volume of solvent (75% DMSO) was injected as a vehicle control. Blood was collected from the retro-orbital venous plexus while animals were under anesthesia. Data on blood electrolytes other than creatinine and magnesium (Mg2þ) were analyzed using the iSTAT EC8þ (Abbott, Inc., Abbott Park, IL). Serum creatinine and magnesium levels were measured by the SRL clinical laboratory service (Tokyo, Japan). Urine samples were collected using a mice urine collection cage (Natsume Seisakusho, Tokyo, Japan). Urine collection began 30 min after a single dose of tacrolimus (3 mg/kg i.p.) and saline (70 ml/g i.p.). Saline was administered to facilitate urine excretion. Water and food was not given to mice during the urine collection period. Data from urine were analyzed using the DRI-CHEM 800V (Fujifilm, Tokyo, Japan). 2.2. Western blotting Semiquantitative immunoblotting was performed according to a previously described protocol [13,14]. Kidneys were homogenized and homogenates were separated via centrifugation. The crude membrane fractions were used for the analysis of protein expression. The primary antibodies used in the present study were as follows: rabbit anti-barttin [12], rabbit anti-pNKCC2 [15], rabbit anti-tNKCC2 (Cell Signaling Technology, Inc. #38436S), rabbit antipNCC (S71) [16], rabbit anti-tNCC [17] and rabbit anti-actin (Cytoskeleton, Inc. Denver, CO. AAN01, Lot 121). Alkaline phosphataseconjugated anti-IgG antibodies (Promega Corporation, Fitchburg, WI) were used as secondary antibodies. Western Blue® stabilized substrate (Promega Corporation, Fitchburg, WI) was used to detect the immunoblot signals. The relative intensities of immunoblot bands were analyzed and quantified using ImageJ software
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(National Institutes of Health, Bethesda, MD). 2.3. Statistical methods Data are expressed as means ± SE. A two-way ANOVA was used to identify differences among treatment groups. Bonferroni post hoc test was performed to identify individual differences. Other data was analyzed using an unpaired t-test. For all analyses, P < 0.05 was considered statistically significant. 3. Results 3.1. In Bsndneo/neo mice, levels of phosphorylated NKCC2 and NCC were increased after treatment with tacrolimus Because tacrolimus have been reported to increase phosphorylation of NKCC2 and NCC in WT mice, we first measured levels of phosphorylated NKCC2 and NCC after treatment with tacrolimus in Bsndneo/neo mice. Tacrolimus was administered subcutaneously for 1 week in Bsndneo/neo mice, and then kidneys were analyzed following euthanization. Administration of tacrolimus significantly increased the overall levels and levels of phosphorylated NKCC2 in Bsndneo/neo mice (Fig. 1). Although there was not significant increase in overall expression of NCC compared to the control group, phosphorylation of NCC was significantly increased in tacrolimus treated group of Bsndneo/neo mice. 3.2. Tacrolimus improved serum Kþ levels in Bsndneo/neo mice Hypokalemia and metabolic alkalosis are two of the symptoms displayed by Bsndneo/neo mice in previously published reports [12]. We measured blood electrolytes to determine whether tacrolimus altered levels of Kþ and bicarbonate (HCO3) in the blood. Treatment with tacrolimus for one week significantly attenuated hypokalemia in Bsndneo/neo mice (Table 1). We also evaluated serum Kþ level before and after treatment with tacrolimus, and we confirmed that tacrolimus treatment significantly increases serum Kþ levels (Fig. 2). However, treatment with tacrolimus did not significantly alter levels of serum HCO 3 (Table 1). 3.3. Tacrolimus administration reduced urinary Kþ excretion in Bsndneo/neo mice We evaluated the change in urinary Kþ excretion after a single intraperitoneal administration of tacrolimus in Bsndneo/neo mice. Because almost no excretion of urine was observed during first 30 min after tacrolimus administration, urine collection started 30 min after the administration. Urinary Kþ excretion was significantly decreased 120 min after tacrolimus administration compared to the vehicle group (Fig. 3). Urine volume, Naþ and Cl excretion also tended to decrease after tacrolimus administration. 3.4. Tacrolimus treatment did not impair kidney function but did produce hypomagnesemia Because calcineurin inhibitors may produce kidney damage and hypomagnesemia [18], we evaluated kidney function and serum Mg2þ levels after one week of treatment with tacrolimus. Tacrolimus caused no significant increase in serum creatinine and blood urea nitrogen (BUN) in WT nor Bsndneo/neo mice (Table 2). This result indicated that the increase in serum Kþ levels after tacrolimus treatment was not due to kidney damage. Tacrolimus treatment yielded a significant decrease in serum Mg2þ levels in WT and Bsndneo/neo mice (Table 2).
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Fig. 1. Representative immunoblots and densitometric analysis after tacrolimus treatment. Tacrolimus (2 mg/kg s.c.) was administered to Bsndneo/neo mice for 1 week. The phosphorylation of NCC and NKCC2 increased in the tacrolimus-treated administered group. All values are expressed as means ± SE. *P < 0.05 vs. tacrolimus; n ¼ 6. Abbreviation: n.s., not significant.
Table 1 Results of body weight and blood test after one week tacrolimus treatment in Bsndneo/neo mice. Values represent means ± SE. t-tests were performed to analyze statistical differences between groups. *P < 0.05. Abbreviations: BW, body weight þ (g); Cl, chloride (mmol/l); HCO 3 , bicarbonate ion (mmol/l); K , potassium (mmol/ l); Naþ, sodium (mmol/l).
BW Naþ Kþ Cl HCO3
Vehicle
n
Tacrolimus
n
22.8 ± 0.9 147.0 ± 0.4 3.9 ± 0.1 103.2 ± 0.5 28.5 ± 0.6
10 10 10 10 10
22.9 ± 1.0 148.4 ± 0.5* 4.3 ± 0.1* 104.8 ± 0.9 29.2 ± 0.7
10 10 10 10 10
4. Discussion BS and Gitelman syndrome (GS) are hereditary tubular diseases characterized by salt-losing polyuria, hypokalemia, and metabolic alkalosis. Five types of BS have been identified according to the affected proteins within the TAL [19]. The cause of GS has been identified as mutation in the gene encoding for the NCC [20]. These diseases are caused by impaired Naþ reabsorption in the TAL and DCT. In this study, we demonstrated that tacrolimus partially ameliorated the phenotype of type 4 BS by increasing phosphorylation of both NKCC2 and NCC.
Fig. 2. Changes in serum potassium (Kþ) level before and after one week of treatment with tacrolimus in Bsndneo/neo mice. There was no significant change in serum Kþ levels in a vehicle group, but a significant increase in serum Kþ levels was observed in the tacrolimus-treated group. *P < 0.05; n ¼ 10.
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Fig. 3. Urine excretion tests with tacrolimus administration. Urine was collected every 30 min after a single dose of tacrolimus (3 mg/kg i.p.). Arrows show time points when tacrolimus was administered. The cumulative volume of urine and urine electrolyte excretion were analyzed. In the tacrolimus-treated group, urinary Kþ excretion was significantly decreased. Urine volume and urinary excretion of Naþ and Cl, although not significant, tended to be suppressed.*P < 0.05 vs. tacrolimus; n ¼ 7. Abbreviation: n.s., not significant.
Calcineurin inhibitors used as immunosuppressive drugs in a clinical setting. In previous studies, calcineurin inhibitors were reported to increase the phosphorylation of NCC, which causes salt retention and hyperkalemia [6,7,10]. Although some studies reported that calcineurin inhibitor increased phosphorylation of NKCC2 [9e11], other reports offered contradicting results [6]. Hoorn et al. concluded that the salt retention observed after treatment with calcineurin inhibitors was caused by the increase in NCC phosphorylation in NCC genome-modified mice [6]. Blankenstein et al. concluded that a calcineurin inhibitor directly increased NCC phosphorylation, but the increase in NKCC2 phosphorylation was caused by hormonal effects on arginine vasopressin (AVP) [10]. In our present study, tacrolimus increased phosphorylation both NKCC2 and NCC in Bsndneo/neo mice, which would be beneficial for the treatment of type 4 BS. Hoorn et al. reported that tacrolimus did not cause salt-sensitive hypertension and hyperkalemia in NCC knockout mice [6]. This result indicates that tacrolimus treatment would not be effective in GS patients who lack NCC. Bsndneo/neo mice, which are used in the present study, express very low levels of barttin and ClCeK, and
Table 2 Results of kidney function and serum magnesium level after 1 week of tacrolimus treatment. Two-way ANOVA and Bonferroni test were used to compare the multiple groups. *P < 0.05, Tacrolimus group compared with vehicle group in the same genotype. yP < 0.05, WT compared with Bsndneo/neo mice in the vehicle group. Abbreviations: BUN, blood urea nitrogen (mg/dl); Cr, creatinine (mg/dl); Mg2þ, magnesium (mg/dl).
Vehicle BUN Cr Mg2þ
25.0 ± 1.1 0.19 ± 0.02 2.1 ± 0.1
Funding
Bsndneo/neo
WT n 12 4 4
Tacrolimus 23.9 ± 0.8 0.20 ± 0.02 1.7 ± 0.1*
n 12 5 5
Vehicle y
29.9 ± 2.3 0.22 ± 0.02 2.7 ± 0.2y
they also express NCC and NKCC2 [12]. This result suggested that tacrolimus could be effective in cases of BS and GS that are caused by incomplete deletion of disease-causing proteins. Although some cases of BS with a complete deletion of barttin were reported [1], most of cases of type 4 BS are caused by point mutations, which cause produce a partial, but not total, loss of function in barttin [1,2,21e25]. In some cases of GS, point mutations were most frequently observed in NCC [26]. Therefore, it is possible that tacrolimus could be effective not only in treating type 4 BS, but also other types of BS and GS. Calcineurin inhibitors were reported to decrease levels of serum Mg2þ. In this study, tacrolimus treatment also produced hypomagnesemia (Table 2). Because hypomagnesemia is present in some cases of BS and GS [19,27], the careful monitoring of serum Mg2þ levels should be required during treatment with a calcineurin inhibitor. In addition, calcineurin inhibiters may cause kidney damage as a side-effect. In this study, tacrolimus treatment did not cause kidney damage. However, because NSAIDs are sometimes used for BS, careful monitoring of renal function would be necessary if the patient is administered calcineurin inhibitors and NSAIDs. In summary, administration of tacrolimus enhanced phosphorylation of NCC and NKCC2 and ameliorated hypokalemia in type 4 BS model mice. Tacrolimus may be effective for the treatment of BS and GS.
n
Tacrolimus
n
10 4 4
33.2 ± 2.3 0.22 ± 0.01 1.7 ± 0.1*
10 4 4
This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) from Japan Society of the Promotion of Science (JSPS) [Grant Numbers JP18K19534, JP19H01049, JP19H03556, JP19H03672, JP18K15995, and JP18K15970].
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Tacrolimus ameliorates the phenotypes of type 4 Bartter syndrome model mice through activation of sodiumepotassiume2 chloride cotransporter and sodiumechloride cotransporter Yoshiaki Matsuura, Naohiro Nomura*, Wakana Shoda, Takayasu Mori, Kiyoshi Isobe, Koichiro Susa, Fumiaki Ando, Eisei Sohara, Tatemitsu Rai, Shinichi Uchida Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo, 1138519, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 July 2019 Accepted 22 July 2019 Available online 27 July 2019
Type 4 Bartter syndrome (BS) is caused by genetic mutations in barttin, which is coded for by BSND. Barttin serves as the b-subunit of the ClCeK chloride (Cl) channel, which is widely expressed in distal nephrons. Type 4 BS is characterized by severely impaired reabsorption of salt, which may cause polyuria, hypokalemia, and metabolic alkalosis. Calcineurin inhibitors reportedly induce renal salt retention and hyperkalemia by enhancing the phosphorylation of the sodium (Naþ)epotassium (Kþ)e2Cl cotransporter (NKCC2) and NaþeCl cotransporter (NCC). In addition, we have previously reported that tacrolimus, a calcineurin inhibitor, increases the levels of phosphorylated NCC. In this study, we administered tacrolimus to barttin hypomorphic (Bsndneo/neo) mice, a murine model of type 4 BS that exhibits polyuria, hypokalemia, and metabolic alkalosis. Administration of tacrolimus increased the serum Kþ level and suppressed urinary Kþ excretion. Furthermore, after treatment with tacrolimus, Bsndneo/neo mice increased levels of phosphorylated NCC and NKCC2. We conclude that tacrolimus partially improves clinical phenotypes of Bsndneo/neo mice, and that calcineurin inhibitors might be effective for treating type 4 BS. © 2019 Elsevier Inc. All rights reserved.
Keywords: Bartter syndrome Calcineurin inhibitor Hypokalemia Sodiumechloride cotransporter
1. Introduction Type 4 Bartter syndrome (BS) is characterized by very severe symptoms, including fetal polyuria, which causes maternal polyhydramnios; postnatal polyuria, which causes dehydration; hypokalemic metabolic alkalosis, and sensorineural deafness. Type 4 BS is caused by mutation of barttin (coded by BSND), which is the bsubunit of the ClCeKa/1 and ClCeKb/2 channels [1,2]. Because barttin is widely expressed from the thin ascending loop of Henle to the collecting duct, mutations in BSND lead to severe impairments in renal function. The thick ascending loop of Henle (TAL) and the distal convoluted tubule (DCT) are important for regulating the urinary excretion of electrolytes in the nephron. In the TAL and DCT, sodium (Naþ) and chloride (Cl) are reabsorbed through the Naþepotassium (Kþ)e2Cl cotransporter (NKCC2) and NaþeCl cotransporter (NCC) in conjunction with ClCeKb/2 and barttin. One
* Corresponding author. E-mail address: [email protected] (N. Nomura). https://doi.org/10.1016/j.bbrc.2019.07.086 0006-291X/© 2019 Elsevier Inc. All rights reserved.
commonly used treatment for type 4 BS is the administration of Kþ supplements for hypokalemia. In addition to Kþ supplementation, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARB), and Kþ-sparing diuretics are also used to suppress urinary Kþ excretion. Nonsteroidal anti-inflammatory drugs (NSAIDs) are also used to suppress levels of prostaglandins and decrease urinary volume and Naþ excretion in refractory cases of type 4 BS [3,4]. However, there are still problems associated with these treatments. For instance, Kþ supplements are often needed in high doses, ACE inhibitors and ARB are not tolerated in patients hypotension, and NSAIDs may cause gastrointestinal and kidney damage. Although activators of type 4 BS-related transporters, such as NKCC2 or NCC, could be promising drugs, there are currently no commercially available activators of NKCC2 nor NCC. Inhibitors of calcineurin, such as tacrolimus and cyclosporine, are often used as immunosuppressants to prevent rejection against transplanted organs. Previous studies have shown that calcineurin inhibitors may induce hyperkalemia and hypertension as sideeffects [5]. Studies showing that calcineurin inhibitors may affect
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levels of NCC phosphorylation and salt-sensitive hypertension have also been reported. Hoorn et al. have reported that treatment with tacrolimus, a calcineurin inhibitor, significantly increased NCC phosphorylation in the murine and human kidney, thus leading to salt-sensitive hypertension [6]. Tacrolimus inhibits calcineurin by binding to the 12 kDa FK506-binding protein (FKBP12). Mice lacking FKBP12 in the nephron did not show tacrolimus-induced hypertension or increased levels of phosphorylated NCC [7]. We have previously reported that tacrolimus inhibited high- Kþ-induced NCC dephosphorylation during the acute phase of Kþ load [8]. Calcineurin inhibitors have been shown to increase the phosphorylation of NKCC2 in some rodent studies [9e11]. This study tested the hypothesis that tacrolimus would improve the clinical phenotypes of a type 4 BS mouse model by increasing the phosphorylation of NKCC2 and NCC. We used Bsndneo(R8L)/neo(R8L) (Bsndneo/neo) mice as a type 4 BS model, which have a hypomorphic mutation in BSND that causes symptoms of BS such as polyuria, hypokalemia and metabolic alkalosis [12]. 2. Materials and methods 2.1. Animal experiences All experiments were performed according to the Tokyo Medical and Dental University Animal Experiment Guidelines. The protocol was approved by The Animal Care and Use Committee of Tokyo Medical and Dental University. Bsndneo/neo mice (C57BL6 background) were used as a loss-of-function model of type 4 BS. We used mice of both sexes between the ages of 8e16 weeks, and all animals weighed between 16 and 28 g. These parameters were matched in each group. All mice were fed according to the CL-2 diet (Oriental Yeast Co., Tokyo, Japan). Tacrolimus (Toronto Research Chemicals Inc., Toronto, Canada) was dissolved in 75% dimethyl sulfoxide (DMSO) and administered once a day for a period of one week (2 mg/kg/day s.c.). The equivalent volume of solvent (75% DMSO) was injected as a vehicle control. Blood was collected from the retro-orbital venous plexus while animals were under anesthesia. Data on blood electrolytes other than creatinine and magnesium (Mg2þ) were analyzed using the iSTAT EC8þ (Abbott, Inc., Abbott Park, IL). Serum creatinine and magnesium levels were measured by the SRL clinical laboratory service (Tokyo, Japan). Urine samples were collected using a mice urine collection cage (Natsume Seisakusho, Tokyo, Japan). Urine collection began 30 min after a single dose of tacrolimus (3 mg/kg i.p.) and saline (70 ml/g i.p.). Saline was administered to facilitate urine excretion. Water and food was not given to mice during the urine collection period. Data from urine were analyzed using the DRI-CHEM 800V (Fujifilm, Tokyo, Japan). 2.2. Western blotting Semiquantitative immunoblotting was performed according to a previously described protocol [13,14]. Kidneys were homogenized and homogenates were separated via centrifugation. The crude membrane fractions were used for the analysis of protein expression. The primary antibodies used in the present study were as follows: rabbit anti-barttin [12], rabbit anti-pNKCC2 [15], rabbit anti-tNKCC2 (Cell Signaling Technology, Inc. #38436S), rabbit antipNCC (S71) [16], rabbit anti-tNCC [17] and rabbit anti-actin (Cytoskeleton, Inc. Denver, CO. AAN01, Lot 121). Alkaline phosphataseconjugated anti-IgG antibodies (Promega Corporation, Fitchburg, WI) were used as secondary antibodies. Western Blue® stabilized substrate (Promega Corporation, Fitchburg, WI) was used to detect the immunoblot signals. The relative intensities of immunoblot bands were analyzed and quantified using ImageJ software
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(National Institutes of Health, Bethesda, MD). 2.3. Statistical methods Data are expressed as means ± SE. A two-way ANOVA was used to identify differences among treatment groups. Bonferroni post hoc test was performed to identify individual differences. Other data was analyzed using an unpaired t-test. For all analyses, P < 0.05 was considered statistically significant. 3. Results 3.1. In Bsndneo/neo mice, levels of phosphorylated NKCC2 and NCC were increased after treatment with tacrolimus Because tacrolimus have been reported to increase phosphorylation of NKCC2 and NCC in WT mice, we first measured levels of phosphorylated NKCC2 and NCC after treatment with tacrolimus in Bsndneo/neo mice. Tacrolimus was administered subcutaneously for 1 week in Bsndneo/neo mice, and then kidneys were analyzed following euthanization. Administration of tacrolimus significantly increased the overall levels and levels of phosphorylated NKCC2 in Bsndneo/neo mice (Fig. 1). Although there was not significant increase in overall expression of NCC compared to the control group, phosphorylation of NCC was significantly increased in tacrolimus treated group of Bsndneo/neo mice. 3.2. Tacrolimus improved serum Kþ levels in Bsndneo/neo mice Hypokalemia and metabolic alkalosis are two of the symptoms displayed by Bsndneo/neo mice in previously published reports [12]. We measured blood electrolytes to determine whether tacrolimus altered levels of Kþ and bicarbonate (HCO3) in the blood. Treatment with tacrolimus for one week significantly attenuated hypokalemia in Bsndneo/neo mice (Table 1). We also evaluated serum Kþ level before and after treatment with tacrolimus, and we confirmed that tacrolimus treatment significantly increases serum Kþ levels (Fig. 2). However, treatment with tacrolimus did not significantly alter levels of serum HCO 3 (Table 1). 3.3. Tacrolimus administration reduced urinary Kþ excretion in Bsndneo/neo mice We evaluated the change in urinary Kþ excretion after a single intraperitoneal administration of tacrolimus in Bsndneo/neo mice. Because almost no excretion of urine was observed during first 30 min after tacrolimus administration, urine collection started 30 min after the administration. Urinary Kþ excretion was significantly decreased 120 min after tacrolimus administration compared to the vehicle group (Fig. 3). Urine volume, Naþ and Cl excretion also tended to decrease after tacrolimus administration. 3.4. Tacrolimus treatment did not impair kidney function but did produce hypomagnesemia Because calcineurin inhibitors may produce kidney damage and hypomagnesemia [18], we evaluated kidney function and serum Mg2þ levels after one week of treatment with tacrolimus. Tacrolimus caused no significant increase in serum creatinine and blood urea nitrogen (BUN) in WT nor Bsndneo/neo mice (Table 2). This result indicated that the increase in serum Kþ levels after tacrolimus treatment was not due to kidney damage. Tacrolimus treatment yielded a significant decrease in serum Mg2þ levels in WT and Bsndneo/neo mice (Table 2).
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Fig. 1. Representative immunoblots and densitometric analysis after tacrolimus treatment. Tacrolimus (2 mg/kg s.c.) was administered to Bsndneo/neo mice for 1 week. The phosphorylation of NCC and NKCC2 increased in the tacrolimus-treated administered group. All values are expressed as means ± SE. *P < 0.05 vs. tacrolimus; n ¼ 6. Abbreviation: n.s., not significant.
Table 1 Results of body weight and blood test after one week tacrolimus treatment in Bsndneo/neo mice. Values represent means ± SE. t-tests were performed to analyze statistical differences between groups. *P < 0.05. Abbreviations: BW, body weight þ (g); Cl, chloride (mmol/l); HCO 3 , bicarbonate ion (mmol/l); K , potassium (mmol/ l); Naþ, sodium (mmol/l).
BW Naþ Kþ Cl HCO3
Vehicle
n
Tacrolimus
n
22.8 ± 0.9 147.0 ± 0.4 3.9 ± 0.1 103.2 ± 0.5 28.5 ± 0.6
10 10 10 10 10
22.9 ± 1.0 148.4 ± 0.5* 4.3 ± 0.1* 104.8 ± 0.9 29.2 ± 0.7
10 10 10 10 10
4. Discussion BS and Gitelman syndrome (GS) are hereditary tubular diseases characterized by salt-losing polyuria, hypokalemia, and metabolic alkalosis. Five types of BS have been identified according to the affected proteins within the TAL [19]. The cause of GS has been identified as mutation in the gene encoding for the NCC [20]. These diseases are caused by impaired Naþ reabsorption in the TAL and DCT. In this study, we demonstrated that tacrolimus partially ameliorated the phenotype of type 4 BS by increasing phosphorylation of both NKCC2 and NCC.
Fig. 2. Changes in serum potassium (Kþ) level before and after one week of treatment with tacrolimus in Bsndneo/neo mice. There was no significant change in serum Kþ levels in a vehicle group, but a significant increase in serum Kþ levels was observed in the tacrolimus-treated group. *P < 0.05; n ¼ 10.
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Fig. 3. Urine excretion tests with tacrolimus administration. Urine was collected every 30 min after a single dose of tacrolimus (3 mg/kg i.p.). Arrows show time points when tacrolimus was administered. The cumulative volume of urine and urine electrolyte excretion were analyzed. In the tacrolimus-treated group, urinary Kþ excretion was significantly decreased. Urine volume and urinary excretion of Naþ and Cl, although not significant, tended to be suppressed.*P < 0.05 vs. tacrolimus; n ¼ 7. Abbreviation: n.s., not significant.
Calcineurin inhibitors used as immunosuppressive drugs in a clinical setting. In previous studies, calcineurin inhibitors were reported to increase the phosphorylation of NCC, which causes salt retention and hyperkalemia [6,7,10]. Although some studies reported that calcineurin inhibitor increased phosphorylation of NKCC2 [9e11], other reports offered contradicting results [6]. Hoorn et al. concluded that the salt retention observed after treatment with calcineurin inhibitors was caused by the increase in NCC phosphorylation in NCC genome-modified mice [6]. Blankenstein et al. concluded that a calcineurin inhibitor directly increased NCC phosphorylation, but the increase in NKCC2 phosphorylation was caused by hormonal effects on arginine vasopressin (AVP) [10]. In our present study, tacrolimus increased phosphorylation both NKCC2 and NCC in Bsndneo/neo mice, which would be beneficial for the treatment of type 4 BS. Hoorn et al. reported that tacrolimus did not cause salt-sensitive hypertension and hyperkalemia in NCC knockout mice [6]. This result indicates that tacrolimus treatment would not be effective in GS patients who lack NCC. Bsndneo/neo mice, which are used in the present study, express very low levels of barttin and ClCeK, and
Table 2 Results of kidney function and serum magnesium level after 1 week of tacrolimus treatment. Two-way ANOVA and Bonferroni test were used to compare the multiple groups. *P < 0.05, Tacrolimus group compared with vehicle group in the same genotype. yP < 0.05, WT compared with Bsndneo/neo mice in the vehicle group. Abbreviations: BUN, blood urea nitrogen (mg/dl); Cr, creatinine (mg/dl); Mg2þ, magnesium (mg/dl).
Vehicle BUN Cr Mg2þ
25.0 ± 1.1 0.19 ± 0.02 2.1 ± 0.1
Funding
Bsndneo/neo
WT n 12 4 4
Tacrolimus 23.9 ± 0.8 0.20 ± 0.02 1.7 ± 0.1*
n 12 5 5
Vehicle y
29.9 ± 2.3 0.22 ± 0.02 2.7 ± 0.2y
they also express NCC and NKCC2 [12]. This result suggested that tacrolimus could be effective in cases of BS and GS that are caused by incomplete deletion of disease-causing proteins. Although some cases of BS with a complete deletion of barttin were reported [1], most of cases of type 4 BS are caused by point mutations, which cause produce a partial, but not total, loss of function in barttin [1,2,21e25]. In some cases of GS, point mutations were most frequently observed in NCC [26]. Therefore, it is possible that tacrolimus could be effective not only in treating type 4 BS, but also other types of BS and GS. Calcineurin inhibitors were reported to decrease levels of serum Mg2þ. In this study, tacrolimus treatment also produced hypomagnesemia (Table 2). Because hypomagnesemia is present in some cases of BS and GS [19,27], the careful monitoring of serum Mg2þ levels should be required during treatment with a calcineurin inhibitor. In addition, calcineurin inhibiters may cause kidney damage as a side-effect. In this study, tacrolimus treatment did not cause kidney damage. However, because NSAIDs are sometimes used for BS, careful monitoring of renal function would be necessary if the patient is administered calcineurin inhibitors and NSAIDs. In summary, administration of tacrolimus enhanced phosphorylation of NCC and NKCC2 and ameliorated hypokalemia in type 4 BS model mice. Tacrolimus may be effective for the treatment of BS and GS.
n
Tacrolimus
n
10 4 4
33.2 ± 2.3 0.22 ± 0.01 1.7 ± 0.1*
10 4 4
This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) from Japan Society of the Promotion of Science (JSPS) [Grant Numbers JP18K19534, JP19H01049, JP19H03556, JP19H03672, JP18K15995, and JP18K15970].
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