Volume 55, Issue 2, Pages 454-464 (February 1999) Molecular mechanisms of glucose action on angiotensinogen gene expression in rat proximal tubular cells  Shao-LING Zhang, Janos G. Filep, Thomas C. Hohman, Shiow-SHIH Tang, Julie R. Ingelfinger, John S.D. Chan  Kidney International  Volume 55, Issue 2, Pages 454-464 (February 1999) DOI: 10.1046/j.1523-1755.1999.00271.x Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 1 Effect of D(+)-glucose on the expression of rat angiotensinogen (ANG) in immortalized rat proximal tubule cells (IRPTCs). Cells were incubated to 24hours in the presence of various concentrations of d(+)-glucose. Media were collected and assayed for immunoreactive rat ANG (IR-rANG). The concentration of IR-rANG in the medium containing low d(+)-glucose (5mm; that is, 2.75 ± 0.05ng/ml) was considered as the control level (100%). Each point represents the mean ± sd of three dishes (**P ≤ 0.01 and ***P ≤ 0.005). Similar results were obtained from three independent experiments. Kidney International 1999 55, 454-464DOI: (10.1046/j.1523-1755.1999.00271.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 2 Effect of D(+)-glucose, D-mannitol, L-glucose, and 2-deoxy D(+)-glucose on the expression of rat ANG in IRPTCs. Cells were incubated for 24hours in the presence of low (5mm) or high (25mm) concentration of d(+)-glucose, d-mannitol, l-glucose, and 2-deoxy d(+)-glucose. Media were harvested after 24hours of incubation and were assayed for IR-rANG. The concentration of IR-rANG in the medium containing 5mm d(+)-glucose was considered as the control level (that is, 100%). Each point represents the mean ± sd of three dishes (***P ≤ 0.005). Similar results were obtained from three other experiments. Kidney International 1999 55, 454-464DOI: (10.1046/j.1523-1755.1999.00271.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 3 Inhibitory effect of Tolrestat on the expression of rat ANG in IRPTCs cultured in the presence of 25mM D(+)-glucose. Cells were incubated for 24hours in the presence of 5mm or 25mm d(+)-glucose in the absence or presence of Tolrestat. Media were harvested and assayed for the level of IR-rANG. The levels of IR-rANG in the medium containing low d(+)-glucose (5mm; that is, 3.25 ± 0.05ng/ml) were considered as the controls (100%). The inhibitory effect of Tolrestat is compared with cells that were incubated in 25mm d(+)-glucose in the absence of Tolrestat. Each point represents the mean ± sd of three dishes (*P ≤ 0.05, **P ≤ 0.01, and NS). Similar results were obtained from three other experiments. Kidney International 1999 55, 454-464DOI: (10.1046/j.1523-1755.1999.00271.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 4 Inhibitory effect of staurosporine or H-7 on the expression of rat ANG in IRPTCs stimulated by 25mM D(+)-glucose. Cells were incubated for 24hours in the presence of 5mm or 25mm d(+)-glucose in the absence or presence of staurosporine (A) or H-7 (B). The levels of IR-rANG in the medium containing low d(+)-glucose (5mm) in A (3.69 ± 0.11ng/ml) or in B (3.49 ± 0.08ng/ml) were considered as control (100%). The inhibitory effect of staurosporine or H-7 is compared with cells that were incubated in 25mm d(+)-glucose (in the absence of staurosporine or H-7). Each point represents the mean ± sd of three dishes (**P ≤ 0.01 and ***P ≤ 0.005). Similar results were obtained from three other experiments. Kidney International 1999 55, 454-464DOI: (10.1046/j.1523-1755.1999.00271.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 5 Stimulatory effect of phorbol 12-myristate 13-acetate (PMA) on the expression of rat ANG in IRPTCs cultured in the presence of 25mM D(+)-glucose. Cells were incubated for 24hours in the presence of 5mm or 25mm d(+)-glucose in the absence or presence of PMA. Media were harvested and assayed for the IR-rANG. Levels of IR-rANG in the medium containing low d(+)-glucose (5mm; 3.15 ± 0.05ng/ml) in the absence of PMA were considered as control (100%). The stimulatory effect of PMA is compared with cells that were stimulated by 25mm d(+)-glucose. Each point represents the mean ± sd of three dishes (**P ≤ 0.01 and ***P ≤ 0.005). Similar results were obtained from two other experiments. Kidney International 1999 55, 454-464DOI: (10.1046/j.1523-1755.1999.00271.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 6 Effect of D(+)-glucose on the expression of rANG in IRPTCs preincubated with a high level of PMA. Cells were preincubated for 24hours with 5mm d(+)-glucose (A) or 5mm d(+)-glucose and 10-5 m PMA (B). Then the media were replaced with fresh media containing 5mm d(+)-glucose, 25mm d(+)-glucose, or 25mm d(+)-glucose plus 10-7 PMA and were incubated for an additional 24hours. Then the media were harvested and assayed for immunoreactive rANG (IR-rANG). The concentration of IR-rANG in the medium containing 5mm d(+)-glucose in panels A and B (1.86 ± 0.12ng/ml and 4.16 ± 0.72ng/ml, respectively) were considered as the controls (100%). Each point represents the mean ± sd of three dishes (*P ≤ 0.05 and ***P ≤ 0.005). Similar results were obtained from another experiment. Kidney International 1999 55, 454-464DOI: (10.1046/j.1523-1755.1999.00271.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 7 Effect of PMA on the expression of rat ANG in IRPTCs in the presence of 25mM D(+)-glucose and Tolrestat. Cells were incubated for 24hours in the presence of 5mm d(+)-glucose, 25mm d(+)-glucose, or 25mm d(+)-glucose plus 10-4 m Tolrestat in the absence or presence of various concentrations of PMA (10-11 to 10-7 m). Media were harvested and assayed for the level of IR-rANG. Levels of IR-rANG in the medium containing low d(+)-glucose (5mm; 4.15 ± 0.1ng/ml) in the absence of Tolrestat or PMA were considered as the controls (100%). The effect of PMA is compared with cells that were incubated in the presence of 25mm d(+)-glucose and 10-4 Tolrestat. Each point represents the mean ± sd of three dishes (**P ≤ 0.01). Similar results were obtained from another experiment. Kidney International 1999 55, 454-464DOI: (10.1046/j.1523-1755.1999.00271.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 8 Effect of D(+)-glucose and Tolrestat on the cellular levels of myoinositol, sorbitol, and fructose in IRPTCs. Cells were incubated for 24hours in the presence of 5mm d(+)-glucose, 25mm d(+)-glucose, or 25mm d(+)-glucose plus Tolrestat (10-4 m). Cells were harvested after 24hours of incubation and assayed for sorbitol, fructose, and myoinositol. The levels of myoinositol (93.74 ± 2.32nmol/mg), sorbitol (0.08 ± 0.003nmol/mg), and fructose (0.248 ± 0.06nmol/mg) in the cells incubated in low d(+)-glucose (5mm) in the absence of Tolrestat were considered as the controls (100%). Each point represents the mean ± sd of five dishes of cells (***P ≤ 0.005). Symbols are: (▪) control, 5mm glucose; (□) cells incubated in 25mm d(+)-glucose; (▨) cells incubated in 25mm d(+)-glucose plus 10-4 Tolrestat. Similar results were obtained from another experiment. Kidney International 1999 55, 454-464DOI: (10.1046/j.1523-1755.1999.00271.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 9 Effect of D(+)-glucose and Tolrestat on the cellular levels of diacyglycerol (DAG) in IRPTCs. After a 30-minute incubation in media with 5mm d(+)-glucose, 25mm d(+)-glucose, or 25mm D(+)-glucose plus Tolrestat (10-4 m). Cells were harvested and assayed for DAG levels. The levels of DAG activity in the cells are expressed as nanomol per mg of protein. Each point represents the mean ± sd of six determinations (*P ≤ 0.05). Similar results were obtained from another experiment. Kidney International 1999 55, 454-464DOI: (10.1046/j.1523-1755.1999.00271.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 10 Effect of D(+)-glucose and Tolrestat on the protein kinase C (PKC) activity in IRPTCs. After 60minutes of incubation in media with 5mm d(+)-glucose, 25mm d(+)-glucose, or 25mm d(+)-glucose plus Tolrestat (10-4 m), cells were harvested, and cytosol and membrane fractions were separated and assayed for PKC activity. The levels of PKC activity were expressed as picomol of 32P-ATP incorporated per minute per mg protein. The effect of 25mm glucose, 25mm glucose plus Tolrestat (10-4 m) is compared to the 5mm glucose (control). Each point represents the mean ± sd of six determinations (*P ≤ 0.05). Similar results were obtained from another experiment. Kidney International 1999 55, 454-464DOI: (10.1046/j.1523-1755.1999.00271.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 11 Effect of D(+)-glucose and Tolrestat on the expression of rat ANG mRNA in IRPTCs. After a 24-hour incubation in media with 5mm d(+)-glucose, 25mm d(+)-glucose, or 25mm plus Tolrestat (10-4 m), cells were harvested and assayed for ANG mRNA levels with a RT-PCR assay. The DNA fragments of the RT-PCR reaction mixture were separated on a 1.5% agarose gel and were then transferred onto a nitrocellulose membrane. Subsequently, the membrane was blotted with a 32P-labeled oligonucleotide corresponding to the nucleotides N+944 to N+1020 of rat ANG and N+418 to N+448 of rat G-3-PDH, respectively. The relative densities of the PCR band of ANG (629bp DNA fragment) were compared with the G-3-PDH control (523bp DNA fragment; A). The level of ANG mRNA in the cells incubated in low d(+)-glucose (5mm; that is, a ratio of 0.83 ± 0.17 unit: ANG/G-3-PDH), in the absence of Tolrestat was considered as the control (100%; B). Each point represents the mean ± sd of three dishes (*P ≤ 0.05). Similar results were obtained from another experiment. Kidney International 1999 55, 454-464DOI: (10.1046/j.1523-1755.1999.00271.x) Copyright © 1999 International Society of Nephrology Terms and Conditions