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Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells
1Department of Medicine, University of Colorado School of Medicine, Denver 80262, USA.
The American Journal of Physiology
|March 1, 1997
Summary
Inner medullary collecting duct cells adapt to hypertonicity via organic osmolytes. While extracellular signal-regulated kinases (ERK) activation is inhibited, Jun NH2-terminal kinase (JNK) and p38 pathways may drive adaptation by regulating osmolyte transporter genes.
Area of Science:
- Nephrology
- Cell Biology
- Molecular Biology
Background:
- Inner medullary collecting duct (IMCD) cells in the kidney adapt to hypertonic environments by synthesizing transporters for organic osmolytes.
- Understanding the signaling pathways involved in this adaptation is crucial for kidney function and disease research.
Purpose of the Study:
- To investigate the activation of mitogen-activated protein (MAP) kinases in IMCD-3 cells under hypertonic conditions.
- To determine the functional role of extracellular signal-regulated kinase 2 (ERK2) in the osmoregulatory response of IMCD cells.
- To elucidate the involvement of Jun NH2-terminal kinase (JNK) and p38 MAP kinase in cellular adaptation to hypertonicity.
Main Methods:
- IMCD-3 cells were exposed to hypertonic media (600 mosmol/kgH2O).
- Cell extracts were fractionated using Mono Q fast-performance liquid chromatography and assayed for MAP kinase activity.
- Western blotting was used to identify JNK, ERK1/2, and p38 MAP kinase.
- The effect of PD-098059 (MEK inhibitor) on ERK activation and inositol uptake was assessed.
- JNK activity was measured by phosphorylation of GST-cJun-(1-79) under varying osmolalities (300-900 mosmol/kgH2O) induced by NaCl, mannitol, or urea.
- Protein kinase C (PKC) involvement was tested by downregulation using phorbol esters.
Main Results:
- Three peaks of MAP kinase activity were identified, corresponding to JNK, ERK1/2, and p38.
- Hypertonicity activated ERK1/2 and p38, but inhibition of ERK activation by PD-098059 did not affect the osmoregulatory increase in inositol uptake.
- JNK activity showed a marked 20- to 40-fold increase with increasing osmolality (NaCl or mannitol), with a prompt response starting at 2 minutes.
- Urea induced a more modest JNK activation.
- PKC downregulation minimally affected the JNK response to hyperosmolality.
Conclusions:
- ERK activation by hypertonicity is not essential for the osmoregulatory increase in inositol transport in IMCD-3 cells.
- JNK and p38 MAP kinase pathways are likely involved in the initial signaling events leading to adaptation.
- These pathways may play a critical role in the transcriptional regulation of organic osmolyte transporter genes in response to extracellular hypertonicity.