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First published online 30 May 2006
doi: 10.1242/jcs.02971

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Loss of calcineurin A results in altered trafficking of AQP2 and in nephrogenic diabetes insipidus

¹ Department of Medicine/Nephrology, Emory University, Atlanta, GA 30322, USA and Atlanta Veterans Affairs Medical Center, Atlanta, GA 30033, USA
² Department of Medicine/Nephrology, University of Texas Health Science Center San Antonio, and South Texas Veterans Health Care System, Audie Murphy Division, San Antonio, TX 78229, USA

View larger version (29K): [in a new window]   Fig. 1. Urine and serum osmolality. (A) Urine osmolality was measured at multiple time points. Data are shown as the mean ± s.e.m. of 4-10 mice per time point. **P<0.001 compared with +/+ mice (ANOVA). (B) Serum osmolality was measured 18-24 days post-natal. Bars show the mean ± s.e.m. of 4-6 mice. *P<0.05 compared with +/+ mice (Student's t-test). (C) Urine osmolalities of +/+, +/-, and -/- mice were measured before and 60 minutes after DDAVP application. Data shown are ten mice per genotype. *P<0.05 compared with +/+ mice (Student's t-test), **P<0.05 (paired t-test).

View larger version (91K): [in a new window]   Fig. 2. Expression of the vasopressin receptor in CnA knockout mice. (A,B) Expression of V2R was visualized by immunohistochemistry in kidney sections of (A) +/+ and (B) -/- mice using the DAB chromagen and counterstaining with hematoxylin. Data are representative of at least four mice. (C) Expression of the V2R was also determined by first immunoprecipitating with a specific antibody against the receptor and then immunoblotting with the same antibody. Lanes 1-3, +/+ mice; lanes 4-6, -/- mice. Bar graph on the right shows expression of V2R as the mean ± s.e.m. ns, not significant.

View larger version (139K): [in a new window]   Fig. 3. Characterization of AQP2 in CnA +/+ and -/- mice. Expression of total AQP2 was visualized by immunohistochemistry in kidney sections of (A) +/+ and (B) -/- mice using the DAB chromagen and counterstaining with hematoxylin. Data are representative of at least four mice. (C) AQP2 expression was determined by western blotting with specific antibodies. Characteristic forms of glycosylated protein (Gly) is observed at 35-45 kDa and non-glycosylated (NG) at 29 kDa. Lanes 1-3, +/+; lanes 4-6, +/-; lanes 7-9, -/-. Expression of actin served as a control. Results from two independent experiments were evaluated by semi-quantitative analysis and results are shown as bar graphs as the mean ± s.e.m.

View larger version (60K): [in a new window]   Fig. 4. Phosphorylation of AQP2 by DDAVP in CnA +/+ and -/- mice. (A) P-AQP2 was detected by immunoblotting with a specific antibody in medullary protein lysates from +/+ mice treated either with vehicle control or DDAVP for 1 hour. Actin was included as a control. Lanes 1-3, untreated; lanes 4-7, plus DDAVP. Results from typical immunoblots were evaluated by semi-quantitative analysis and are shown in bar graph as the mean ± s.e.m. *P<0.05 (Student's t-test). (B) P-AQP2 was detected in medullary protein lysates from -/- mice treated with either control or plus DDAVP for 1 hour. Actin was included as an internal control. Lanes 1-3: control mice; and lanes 4-7: plus DDAVP. Results from typical immunoblots were evaluated by semi-quantitative analysis and results are shown as bar graphs as the mean ± s.e.m. (C) Expression of PKA was determined by western blotting using a specific antibody in +/+, +/- and -/- mouse kidney-cell homogenates. Results from two independent experiments were evaluated by semi-quantitative analysis and are shown as bar graphs as the mean ± s.e.m. (D) In an in vitro assay (see Materials and Methods) activity of PKA was determined in homogenates of whole kidneys of CnA +/+ and -/- mice that had been either treated with vehicle only (control) or with DDAVP for 1 hour. Bars show the mean ± s.e.m. of 4-6 animals per genotype. *P<0.05 compared with +/+ control (Student's t-test). (E) Intracellular localization of AQP2 was examined by immunohistochemistry in the inner strip of the outer medulla in both +/+ and -/- mice following DDAVP treatment. Arrows identify AQP2 apical localization in +/+ and cellular/baso-lateral distribution in -/- mice. Magnification is 40 x. Results of 4-6 mice per group were evaluated by semi-quantitative analysis and are shown in bar graph as the mean ± s.e.m. *P<0.05.

View larger version (203K): [in a new window]   Fig. 5. Subcellular localization of AQP2 in CnA +/+ and -/- mice. Ultrathin IM sections of kidneys from +/+ and -/- mice were immunogold labeled for AQP2, and viewed by electron microscopy. (A) +/+, (B) -/-, (C) +/+ plus DDAVP, (D) -/- plus DDAVP. Bars, 100 nm.

View larger version (56K): [in a new window]   Fig. 6. Expression of total and p-AQP2 in IMCD-cell vesicle and membrane fractions. (A) IMCD-cell vesicle fractions (VFs) were isolated from +/+, +/- and -/- mice. Expression of total and p-AQP2 was detected by immunoblotting with specific antibodies. Actin was included as a control. Lanes 1-3, +/+; 4-6, +/-; and 7-9, -/-. Levels of total and p-AQP2 were evaluated by semi-quantitative analysis, normalized against actin and shown in bar graph. Data show the mean ± s.e.m. of data from 3-5 mice. *P<0.05 (Student's t-test). (B) Calcineurin activity and expression were examined by western blotting VFs with an antibody that crossreacts with the phosphatase domain of all three A subunit isoforms. Actin expression was examined in the same samples as an internal control. (C) Calcineurin activity in isolated VFs was determined in an vitro assay. Bar graphs show the mean ± s.e.m. of 4-6 samples per group. *P<0.01 (Student's t-test). (D) MFs were prepared and expression of total and p-AQP2 were found by direct immunoblotting. Lanes 1-3, +/+; lanes 4-6, +/-; lanes 7-9, -/-. Actin was included as a control. Levels of total and p-AQP2 were evaluated by semi-quantitative analysis and results are shown in bar graphs as the mean ± s.e.m. of data from 3-5 mice. *P<0.05, **P<0.01 (Student's t-test).

View larger version (31K): [in a new window]   Fig. 7. Effect of CsA on calcineurin expression and activity and on response to DDAVP. (A) In an vitro assay (see Materials and Methods), activity of calcineurin was determined in homogenized whole kidneys from control, DDAVP, and CsA+DDAVP mice. *P<0.05 compared with wild-type, **P<0.05 compared with DDAVP-treated mice. Bar graphs show the mean ± s.e.m. (B) Expression of total CnA was determined in protein from homogenized whole kidneys of control, DDAVP and CsA+DDAVP animals by direct immunoblotting with specific antibodies. Actin was included as a control. Each lane contains protein isolated from one animal. Lanes 1-3, control; lanes 4-6, DDAVP; lanes 7-9: CsA + DDAVP. (C) Urine osmolality was measured in control and CsA-treated animals before and 60 minutes after the subcutaneous injection of DDAVP. Data shown are from eight individual animals per treatment group. *P<0.05 (paired t-test). Also shown is the mean osmolality of each group.

View larger version (47K): [in a new window]   Fig. 8. Effect of CsA on DDAVP-mediated regulation of AQP2. (A) Expression of p-AQP2 and total AQP2 was determined in whole kidney lysates of control, DDAVP, and DDAVP + CsA-treated mice by immunoblotting with specific antibodies. Actin was included as a control. Lanes 1-3, control; lanes 4-6, DDAVP; lanes 7-9, CsA + DDAVP. Amounts of total and p-AQP2 were evaluated by semi-quantitative analysis and results are shown in bar graphs. (B) AQP2 localization was detected by immunohistochemistry in kidney IM sections with the DAB chromagen and sections were counterstained with hematoxylin. (i) Control, (ii) DDAVP, (iii) DDAVP + CsA. Shown are typical data of three mice per group. Magnification is 40x Results of immunohistochemistry were evaluated by semi-quantitative analysis and are shown in as the mean ± s.e.m. *P<0.05.

View larger version (44K): [in a new window]   Fig. 9. Effect of CsA on distribution of VF and MF, and phosphorylation of AQP2. (A) IMCD-cell VFs were isolated from control and CsA-treated mice. Expression of total and p-AQP2 was detected by immunoblotting with specific antibodies. Tubulin was included as a control. Lanes 1-3, control; lanes 4-7, CsA-treated mice. Levels of total and p-AQP2 were evaluated by semi-quantitative analysis and data from 3-5 mice are shown as the mean ± s.e.m. *P<0.05 (Student's t-test). (B) MFs were prepared and expression of total and p-AQP2 was detected by direct immunoblotting. Tubulin was included as a control. Lanes 1-4, control; lanes 5-8, CsA-treated. Levels of total and p-AQP2 were evaluated by semi-quantitative analysis and data from 3-5 mice are shown as the mean ± s.e.m. *P<0.05, **P<0.01 (Student's t-test).