Table 1.
Nucleotide sequences of the primers used in this study.
Fig 1.
High glucose augments AGT mRNA level in HK-2 cells in a time- and dose-dependent manner.
(A) AGT mRNA levels measured at different time points in HK-2 cells respectively treated with normal glucose (5.5 mM) and high glucose (15.0 mM). Compared with normal glucose treatment, high glucose significantly augmented AGT mRNA level from 39 h to 63 h. (B) AGT mRNA levels measured in HK-2 cells respectively treated with different glucose concentrations for 48 h. Compared with normal glucose treatment, high glucose augmented AGT mRNA level with the most significant effects by 15.0 or 20.0 mM glucose. (C) The effect of high glucose on AGT mRNA level was not due to osmotic stress which was further balanced with mannitol treatment for 48 h. Data are expressed as relative values to the 0h group (A) or normal glucose group (B and C). Values are presented as mean ± SEM. *P<0.05 vs. 0h group with the same glucose concentration; #P<0.05 vs. normal glucose group with the same treating time. N = 3~6.
Fig 2.
High glucose augments AGT secretion of HK-2 cells.
Levels of secreted AGT in the culture medium of HK-2 cells respectively treated with normal glucose (5.5 mM), high glucose (15.0 mM) and normal glucose plus 9.5 mM mannitol. Compared with normal glucose treatment, high glucose significantly augmented AGT secretion into the medium, which was not affected by mannitol-induced osmotic stress. Data are expressed as relative values normalized by total cellular protein amount in the dish. Values are presented as mean ± SEM. *P<0.05 vs. normal glucose group. N = 3~6.
Fig 3.
High glucose augments AGT promoter activity of HK-2 cells transfected with the promoter sequences constructs.
(A) Consecutive 5’-end deletion mutant constructs of the DNA sequences of human AGT promoter used in this study. Horizontal line represents DNA sequences of the relevant deletion mutant construct. Open box represents the luciferase reporter. (B) Effects of high glucose on AGT promoter activity of HK-2 cells respectively transfected the constructs with 5’-ends from -22 to -4,358. Compared with normal glucose (5.5 mM) treatment, high glucose (15.0 mM) significantly augmented AGT promoter activity of HK-2 cells respectively transfected with the constructs with 5’-ends from -344 to -4,358. Additionally, within high glucose treatment groups, consecutive increase of AGT promoter activity was observed in HK-2 cells harboring constructs with 5’-ends from -344 to -1,896, while followed by marked decrease of AGT promoter activity in HK-2 cells harboring constructs with 5’-ends from -2,414 to -4,358. Data are expressed as relative values to the empty vector transfection group under the same glucose concentration. Values are presented as mean ± SEM. *P<0.05 vs. normal glucose group with the same construct. N = 3~6.
Table 2.
Corresponding transcriptional factors of human AGT promoter sequence region -344 to -1896 (score ≧ 50).
Table 3.
Corresponding transcriptional factors of human AGT promoter sequence region -344 to -1896 (50 > score ≧ 40).
Table 4.
Corresponding transcriptional factors of human AGT promoter sequence region -22 to -344 (score ≧ 20).
Fig 4.
Mutation in HNF-5 binding sites reduces the effects of high glucose on AGT promoter activity as well as AGT mRNA and secretion levels.
(A, B and C) Constructs with respective mutation of HNF-5 (A), CREB (B) or MEF2 (C) binding sites in main human AGT promoter sequences (AGT_-4,358/+122). Within binding sites, the underlined base pairs were substituted by mutagenesis. Horizontal line represents human AGT_-4,358/+122 with relevant mutation. Open box represents the luciferase reporter. (D, E and F) Effects of high glucose treatment on AGT promoter activity in HK-2 cells, which were transfected with either intact or relevant binding sites mutated construct of human AGT_-4,358/+122. Compared with normal glucose (5.5 mM) treatment, high glucose (15.0 mM) significantly augmented AGT promoter activity of HK-2 cells transfected with the intact construct of human AGT_-4,358/+122 (D, E and F). Furthermore, the above effect was abolished in HK-2 cells with mutated HNF-5 binding sites (D), while not affected by the binding sites mutation of CREB (E) or MEF2 (F). g: guanine; a: adenine; c: cytosine; t: thymine. Data are expressed as relative values to the empty vector transfection group under the same glucose concentration. (G) Mutation in HNF-5 binding sites attenuated the effects of high glucose on AGT mRNA level. Data are expressed as relative values to the empty vector transfection group with normal glucose. (H) Mutation in HNF-5 binding sites attenuated the effects of high glucose on AGT secretion level. Values are presented as mean ± SEM. *P<0.05 vs. normal glucose (5.5 mM) group with the same construct; #P<0.05 vs. intact human AGT_-4,358/+122 transfection group under the same glucose concentration; N.S.: no significant difference. N = 3~6.
Fig 5.
Binding level of HNF-5 to AGT promoter sequences is reduced by mutation in its binding sites.
(A and B) Levels of DNA immuno-precipitated by HNF-5 protein from HK-2 cells respectively transfected with empty vector, intact and HNF-5 binding sites mutated main human AGT promoter sequences (AGT_-4,358/+122), detected through band intensities after PCR (A) and quantitative real-time PCR (B). Compared with the group with intact human AGT_-4,358/+122, the binding level of HNF-5 protein to human AGT_-4,358/+122 significantly decreased in HK-2 cells with HNF-5 binding sites mutation. Data of quantitative real-time PCR are expressed as relative values to the empty vector transfection group. DNA (−) indicates the absence of DNA, IP: immuno-precipitation. Values are presented as mean ± SEM. N = 3~6.
Fig 6.
Schematic diagram summarizing the role of HNF-5 in AGT augmentation by high glucose in human RPTCs.
In human RPTCs, through HNF-5, high glucose enhances the activity of AGT gene promoter, thus increases the levels of transcription, translation and secretion of AGT, in result, the levels of AGT mRNA and AGT secretion were augmented. g: guanine; a: adenine; c: cytosine; t: thymine.