Effect of Hydrocortisone on Angiotensinogen (AGT) Mutation–Causing Autosomal Recessive Renal Tubular Dysgenesis

Figures

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  Figure 1. Sanger sequence, cDNA, and amino acids sequence. Sanger sequencing illustrated a large deletion of Angiotensinogen (AGT), E3_E4 del: 2870 bp deletion with 9 bp insertion. With cDNA analysis, this deletion of AGT leads to a truncated protein (292 amino acids).

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  Figure 2. The expression level and stability of various AGT proteins. (A) Human kidney-2 (HK-2) cells (5 × 104 cells/well) were transiently transfected with HA.AGT wild-type (AA 1–485), pSG5.HA.AGT (AA 1–375/R375Q), and pSG5.HA.AGT truncated (AA 1–292). (B,C) HK-2 cells (5 × 104 cells/well) were transiently transfected with 0.4 μg pSG5.HA.AGT wild-type (AA 1–485) and pSG5.HA.AGT (AA 1–375/R375Q) and 0.8 μg pSG5.HA.AGT truncated (AA 1–292). (C) The relative stability was calculated and compared with 0 h cycloheximide (CHX) treatment. The results (A,B) are representative of three independent experiments.

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  Figure 3. Proximity ligation assay between AGT and renin in kidney. (A) Illustration of deleted region of truncated AGT protein involving serpin domain and cleavage sites of renin and angiotensin-converting enzyme (ACE); (B,C) HK-2 cells (5 × 104 cells/well) were transiently transfected with 0.4 μg pSG5.HA.AGT wild-type (AA 1–485) and pSG5.HA.AGT (AA 1–375/R375Q) and 0.8 μg pSG5.HA.AGT truncated (AA 1–292). Measurement of Renin-AGT interaction by proximity ligation assay. The results (C) are representative of three independent experiments.

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  Figure 4. The effect of hydrocortisone on mutant AGT proteins. (A) L02 cells were treated with indicted amount of hydrocortisone and dexamethasone for 24 h; (B) L02 cells were transiently transfected with 0.4 μg pSG5.HA.AGT wild-type (AA 1–485) and pSG5.HA.AGT (AA 1–375/R375Q) and 0.8 μg pSG5.HA.AGT truncated (AA 1–292) and transfected cells were treated with dexamethasone and hydrocortisone for 24 h; (C,D) L02 cells were transiently transfected with pSG5.HA.AGT (1–485 and 1–292) and transfected cells treated with 10 μM hydrocortisone for 46 h and 50 μg/mL CHX for 20, 40, 80, 180, and 360 min. The relative stabilities of wild-type and mutant AGT proteins were calculated and compared with 0 h CHX treatment. Both wild-type and truncated AGTs increased with a decreased glucocorticoid receptor (GR) after hydrocortisone treatment. (E) Effect of proteasome inhibitor on AGT degradation. L02 cells were transiently transfected with 0.4 μg pSG5.HA.AGT wild-type (AA 1–485) and 0.8 μg pSG5.HA.AGT truncated (AA 1–292) and transfected cells treated with 0, 1, 3, 5, and 10 μM MG132 for 2 h and then treated with 10 μg/mL CHX for 120 min. p53 is for a control MG132 treatment and alpha-actinin (ACTN) is for…

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  Figure 5. The effect of hydrocortisone on the interaction between AGT proteins and renin in liver. (A) L02 cells (8 × 104 cells/well) were transiently transfected with 0.4 μg pSG5.HA.AGT wild-type (AA 1–485) and pSG5.HA.AGT (AA 1–375/R375Q) and 0.8 μg pSG5.HA.AGT truncated (AA 1–292) and transfected cells were treated with hydrocortisone for 24 h. (B) Measurement of Renin-AGT interaction by proximity ligation assay. The results are representative of three independent experiments.

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  Figure 6. The functional status of various AGTs examined by the modulation of GR-dependent MMTV-LUC activity in HK-2 cells. (A) HK-2 cells were transfected with 0.25 μg of the MMTV-LUC reporter plasmid and pKSX.GR (0.15 μg) and/or indicated amounts of various AGT expression vectors in the absence or presence of 100 nM Dexamethasone or 1 μM hydrocortisone for 46 h. The presented data are the means of three experiments (mean ± S.D.; n = 3); (B) The mutant AGT exerts different glucocorticoid receptor-dependent transactivation than wild AGT.

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  Figure 7. Proposed Pathogenesis of Angiotensin II deficiency due to large deletion of AGT in Rental Tubular Dysgenesis. Normally, wild-type AGTs are converted to angiotensin II through enzymes of renin and ACE. Mutation in AGT gene yields a shortened and abnormal AGT with decreased cleavage by renin and subsequent low serum angiotensin I and II. Hydrocortisone increases the production of angiotensin II by enhancing the interaction between renin and truncated AGT.