Related Experiment Videos
Effect of second messenger systems on oxalate uptake in renal epithelial cells
L Calò1, T R Wandzilak, P A Davis
1Division of Nephrology, University of Padova, Italy.
Abstract:
The oxalate transport system along with protein phosphorylation appears to be deranged in stone formers. This study was undertaken to characterize in LLC-PK1 cells in culture the effect of altering specific intracellular second messenger systems on oxalate uptake. Cellular uptake experiments were performed at 37 degrees C in buffer [265 mM mannitol, 5 mM NaOH, 5 mM KOH, 10 mM Ca-EGTA, 25 mM HEPES/TRIS, pH = 7.4 or in Hank's balanced salt solution (HBSS)] containing 200 microM labeled oxalate (1-14C, 0.3 microCi). Cells were preincubated with DAG (final concentration of 100 microM), phorbol myristate acetate (10 microM), forskolin (50 microM), 8-bromo-cyclic AMP (50 microM), trifluoroperazine (20 microM) and low molecular weight heparin (1 mg/ml) for 10 min in the presence and absence of the anion transport inhibitor DIDS (100 microM) and the effect(s) on oxalate uptake at 10, 25 and 45 min incubation were determined. Chemicals (DAG, forskolin, TPA and 8-bromo-cAMP) which stimulate protein kinase A or C activity resulted in an increased uptake of oxalate while inhibitors of these systems (trifluoroperazine and low molecular weight heparin) resulted in decreased oxalate uptake. The results demonstrate that oxalate uptake in renal tubular cells is modulated by protein kinase C and A dependent mechanisms.
Insights
Oxalate transport in kidney cells is influenced by intracellular signaling pathways. Stimulating protein kinase A or C increases oxalate uptake, while inhibitors decrease it, revealing key mechanisms in stone formation.
Area of Science:
- Nephrology
- Cell Biology
- Biochemistry
Background:
- Oxalate transport and protein phosphorylation are implicated in kidney stone formation.
- Intracellular second messenger systems may play a role in regulating oxalate transport.
Purpose of the Study:
- To investigate the impact of altering intracellular second messenger systems on oxalate uptake in LLC-PK1 cells.
- To characterize the role of protein kinase A and C in modulating oxalate transport.
Main Methods:
- LLC-PK1 cells were used to study cellular oxalate uptake.
- Cells were incubated with various agents affecting protein kinase A and C activity, including DAG, forskolin, TPA, 8-bromo-cAMP, trifluoroperazine, and heparin.
- Oxalate uptake was measured using radiolabeled oxalate (1-14C) over 45 minutes, with and without the anion transport inhibitor DIDS.
Main Results:
- Chemicals that stimulate protein kinase A or C (DAG, forskolin, TPA, 8-bromo-cAMP) significantly increased oxalate uptake.
- Inhibitors of these kinases (trifluoroperazine, heparin) led to a decrease in oxalate uptake.
- These effects were observed within 10-45 minutes of incubation.
Conclusions:
- Oxalate uptake in renal tubular cells is modulated by protein kinase C and A-dependent mechanisms.
- These findings suggest a potential therapeutic target for kidney stone prevention by modulating these signaling pathways.