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Urinary excretion of epidermal growth factor (EGF) and tammhorsfall protein (THP) in rats with altered kidney function
Abstracts from the l l t h International Symposium on Regulatory Peptides
191
URINARY EXCRETION OF EPIDERMAL GROWTH FACTOR (EGF) AND TAMMH O R S F A L L P R O T E I N ( T H P ) IN R A T S W I T H A L T E R E D K I D N E Y F U N C T I O N . J. Thulesen, P.E. J¢rgensen*, O. Torffvit#, E. Nex¢*, S,S. Poulsen. Institute of Medical Anatomy, University of Copenhagen, Denmark; * Depart. of Clinical Biochemistry, Aarhus University Hospital, Denmark and # Depart. of lntemal Medicine, Lund University Hospital, Sweden. EGF and THP are both synthesized in the thick ascending limb of Henle and in the early part of the distal convoluted tubules. EGF is a mitogenic peptide with a molecular weight of 6kDa and THP is an extremely carbohydrate-rich urinary glycoprotein with a molecular weight of approximately 70,000 kDa. They are both present in urine in high amounts. However, their functional significance has not been well defined. The present study was undertaken to measure these peptides in urine from rats with altered kidney function. The following groups were investigated: [ I ] streptozotocin (60 mg/kg) - diabetic (DIA); [2] insulin-treated streptozotocindiabetic (INS-DIA); [3] rats with non-osmotic polyuria (POL) (food restriction and 5% sucrose in drinking water); [4] unilateral nephrectomized (NEPHR) and; [5] control rats (CTRL). The groups were started on day 0 and diurnal volume of urine was collected on days 1, 7, 14 and 21. The kidneys were weighed and evaluated on day 21 by immunohistochemistry and in-situ hybridization. Renal hypertrophy was observed in DIA and NEPHR. DIA and POL had both significantly polyuria compared to the other groups. The diurnal urinary excretion of EGF was unchanged in DIA and POL, however, both groups had significantly increased content ofTHP (DIA; on days 14 and 21, and POL; on all days). There was a significant increment in the content of THP from day 14 to day 21 in DIA, which might reflect changes in tubular function of the diabetic kidney. INS-DIA had values comparable to CTRL. NEPHR had a significant reduction in the urinary excretion of EGF on all days (day 1: 60%, day 21: 83%) when compared to the CTRL whereas, the excretion of THP was unchanged. Thus, the renal synthesis of both EGF and THP was compensated in unilateral nephrectomy, since a 50% reduction in the urinary content, otherwise, was expected. These findings indicate that the diurnal urinary EGF excretion is rather constant, only dependent on the capacity of the kidneys and completely independent on the urinary volume and thereby, rats with polyuria have very low concentrations of EGF in urine. In contrast with that, the diurnal excretion of THP varied considerably, increasing in parallel with the urinary volume. Thus, the concentrations in the urine are almost constant even in the case of considerable polyuria. Accordingly, the renal excretion of EGF from the remaining kidney after nephrectomy is reduced and seems to increase in parallel with renal hypertrophy while the excretion of THP is unchanged and follows renal workload. In conclusion, the present results imply that the synthesis of EGF and THP is regulated differently even though that both peptides are synthesized at the same renal site.
MOLECULAR MECHANISMS FOR THE GROWTH FACTOR ACTION OF GASTRIN. A. Todisco. Y. Takeuchi, J. Yamada, A. Ummov, V. Stepan, C. J. Dickinson, T. Yamada. U. of Michigan Med. Ctr., Ann Arbor, MI. In previous studies, we have observed that gastrin has a CCKB receptor mediated growth promoting effect on the AR4-2J rat acinar cell line and that this effect is paralleled by induction of expression of the early response gene c-fos. We undertook these experiments to elucidate the mechanism for induction of c-fos and the linkage of this action to the trophic effects of gastrin. Growth factor effects are know to be mediated through a specific region of the c-fos promoter known as the serum response element (SRE). When we examined the effect of gastrin (10-SM) on AR4-2J cells transfected with a SRE-luciferase reporter gene plasmid we observed a six-fold induction in luciferase activity, an effect similar to the induction observed in the presence of EGF (10-aM). This effect of gastrin was blocked by the specific CCK B receptor antagonist D2 but not by pertussis toxin, indicating that gastrin targets the SRE through a mechanism independent of inhibitory G proteins. Inhibition of protein kinase C (PKC) either by prolonged (24h) exposure of the cells to the phorbol ester TPA or by incubation with the selective inhibitor GFI09203X (3.5x10 -6 M) resulted in a 40-50% reduction in luciferase activity. As activation of the cfos SRE is known to require induction of mitogen activated protein kinase (MAPK) and its upstream activator MEK, we examined the effect of the specific MEK inhibitor PD98059 and observed that this compound inhibited gastrin stimulated SRE-luciferase activity by 80%. Thus, both PKC-dependent and PKC-independent, MEKdependent pathways appear to mediate gastrin action. We measured MAPK enzyme activity in AR4-2J cells, via in-gel kinase assays and observed that gastrin (10-7-10-1OM) induced MAPK enzyme activity in a dose dependent manner. In parallel with the results obtained with the luciferase assays, inhibition of PKC and MEK produced, respectively, partial and near total inhibition of gastrin induced MAPK activity. MAPK activation induces c-los gene expression by phosphorylation of the transcription factor EIk-I which binds to the c-fos SRE. To examine if this pathway mediated the effects of gastrin, we used a yeast hybrid system involving co-transfection of the cells with a chimeric GAL4-EIk-1 expression vector and the 5XGAL-luciferase reporter plasmid. In this system GAL4Elk-1 transactivates and stimulates luciferase activity only if the carboxyl-terminus of EIk-I is phosphorylated by MAPK. Gastrin induced a 10-fold induction of 5XGAL-luciferase activity. To confirm the involvement of MEK activation in the growth promoting effect of gastrin, we examined whether PD98059 would inhibit gastrin stimulated AR4-2J cell proliferation, Gastrin (10 -9) stimulated AR4-2J cell proliferation as measured by incorporation of 3H-thymidine and PD98059 (5x10 -5 M) inhibited this response by 60%. Our data led us to conclude that the trophic actions of gastrin are mediated by MAPK induced c-fos gene expression via PKCdependent and PKC-independent pathways.
191
URINARY EXCRETION OF EPIDERMAL GROWTH FACTOR (EGF) AND TAMMH O R S F A L L P R O T E I N ( T H P ) IN R A T S W I T H A L T E R E D K I D N E Y F U N C T I O N . J. Thulesen, P.E. J¢rgensen*, O. Torffvit#, E. Nex¢*, S,S. Poulsen. Institute of Medical Anatomy, University of Copenhagen, Denmark; * Depart. of Clinical Biochemistry, Aarhus University Hospital, Denmark and # Depart. of lntemal Medicine, Lund University Hospital, Sweden. EGF and THP are both synthesized in the thick ascending limb of Henle and in the early part of the distal convoluted tubules. EGF is a mitogenic peptide with a molecular weight of 6kDa and THP is an extremely carbohydrate-rich urinary glycoprotein with a molecular weight of approximately 70,000 kDa. They are both present in urine in high amounts. However, their functional significance has not been well defined. The present study was undertaken to measure these peptides in urine from rats with altered kidney function. The following groups were investigated: [ I ] streptozotocin (60 mg/kg) - diabetic (DIA); [2] insulin-treated streptozotocindiabetic (INS-DIA); [3] rats with non-osmotic polyuria (POL) (food restriction and 5% sucrose in drinking water); [4] unilateral nephrectomized (NEPHR) and; [5] control rats (CTRL). The groups were started on day 0 and diurnal volume of urine was collected on days 1, 7, 14 and 21. The kidneys were weighed and evaluated on day 21 by immunohistochemistry and in-situ hybridization. Renal hypertrophy was observed in DIA and NEPHR. DIA and POL had both significantly polyuria compared to the other groups. The diurnal urinary excretion of EGF was unchanged in DIA and POL, however, both groups had significantly increased content ofTHP (DIA; on days 14 and 21, and POL; on all days). There was a significant increment in the content of THP from day 14 to day 21 in DIA, which might reflect changes in tubular function of the diabetic kidney. INS-DIA had values comparable to CTRL. NEPHR had a significant reduction in the urinary excretion of EGF on all days (day 1: 60%, day 21: 83%) when compared to the CTRL whereas, the excretion of THP was unchanged. Thus, the renal synthesis of both EGF and THP was compensated in unilateral nephrectomy, since a 50% reduction in the urinary content, otherwise, was expected. These findings indicate that the diurnal urinary EGF excretion is rather constant, only dependent on the capacity of the kidneys and completely independent on the urinary volume and thereby, rats with polyuria have very low concentrations of EGF in urine. In contrast with that, the diurnal excretion of THP varied considerably, increasing in parallel with the urinary volume. Thus, the concentrations in the urine are almost constant even in the case of considerable polyuria. Accordingly, the renal excretion of EGF from the remaining kidney after nephrectomy is reduced and seems to increase in parallel with renal hypertrophy while the excretion of THP is unchanged and follows renal workload. In conclusion, the present results imply that the synthesis of EGF and THP is regulated differently even though that both peptides are synthesized at the same renal site.
MOLECULAR MECHANISMS FOR THE GROWTH FACTOR ACTION OF GASTRIN. A. Todisco. Y. Takeuchi, J. Yamada, A. Ummov, V. Stepan, C. J. Dickinson, T. Yamada. U. of Michigan Med. Ctr., Ann Arbor, MI. In previous studies, we have observed that gastrin has a CCKB receptor mediated growth promoting effect on the AR4-2J rat acinar cell line and that this effect is paralleled by induction of expression of the early response gene c-fos. We undertook these experiments to elucidate the mechanism for induction of c-fos and the linkage of this action to the trophic effects of gastrin. Growth factor effects are know to be mediated through a specific region of the c-fos promoter known as the serum response element (SRE). When we examined the effect of gastrin (10-SM) on AR4-2J cells transfected with a SRE-luciferase reporter gene plasmid we observed a six-fold induction in luciferase activity, an effect similar to the induction observed in the presence of EGF (10-aM). This effect of gastrin was blocked by the specific CCK B receptor antagonist D2 but not by pertussis toxin, indicating that gastrin targets the SRE through a mechanism independent of inhibitory G proteins. Inhibition of protein kinase C (PKC) either by prolonged (24h) exposure of the cells to the phorbol ester TPA or by incubation with the selective inhibitor GFI09203X (3.5x10 -6 M) resulted in a 40-50% reduction in luciferase activity. As activation of the cfos SRE is known to require induction of mitogen activated protein kinase (MAPK) and its upstream activator MEK, we examined the effect of the specific MEK inhibitor PD98059 and observed that this compound inhibited gastrin stimulated SRE-luciferase activity by 80%. Thus, both PKC-dependent and PKC-independent, MEKdependent pathways appear to mediate gastrin action. We measured MAPK enzyme activity in AR4-2J cells, via in-gel kinase assays and observed that gastrin (10-7-10-1OM) induced MAPK enzyme activity in a dose dependent manner. In parallel with the results obtained with the luciferase assays, inhibition of PKC and MEK produced, respectively, partial and near total inhibition of gastrin induced MAPK activity. MAPK activation induces c-los gene expression by phosphorylation of the transcription factor EIk-I which binds to the c-fos SRE. To examine if this pathway mediated the effects of gastrin, we used a yeast hybrid system involving co-transfection of the cells with a chimeric GAL4-EIk-1 expression vector and the 5XGAL-luciferase reporter plasmid. In this system GAL4Elk-1 transactivates and stimulates luciferase activity only if the carboxyl-terminus of EIk-I is phosphorylated by MAPK. Gastrin induced a 10-fold induction of 5XGAL-luciferase activity. To confirm the involvement of MEK activation in the growth promoting effect of gastrin, we examined whether PD98059 would inhibit gastrin stimulated AR4-2J cell proliferation, Gastrin (10 -9) stimulated AR4-2J cell proliferation as measured by incorporation of 3H-thymidine and PD98059 (5x10 -5 M) inhibited this response by 60%. Our data led us to conclude that the trophic actions of gastrin are mediated by MAPK induced c-fos gene expression via PKCdependent and PKC-independent pathways.