High glucose concentration stimulates NHE-1 activity in distal nephron cells: the role of the Mek/Erk1/2/p90RSK and p38MAPK signaling pathways.

AIMS

In models of diabetes, distal nephron cells contribute to glucose uptake and oxidation. How these cells contribute to the use of glucose for the regulation of H(+) extrusion remains unknown. We used Madin-Darby Canine Kidney (MDCK) cells to investigate the effect of acute or chronic high glucose concentration on the abundance and activity of the Na(+)/H(+) exchanger (NHE-1).

METHODS

Using RT-PCR, we also evaluated the mRNA expression for sodium glucose co-transporters SGLT1 and SGLT2. Protein abundance was analyzed using immunoblotting, and intracellular pH (pHi) recovery was evaluated using microscopy in conjunction with the fluorescent probe BCECF/AM. The Na(+)-dependent pHi recovery rate was monitored with HOE-694 (50 µM) and/or S3226 (10 µM), specific NHE-1 and NHE-3 inhibitors.

RESULTS

MDCK cells did not express the mRNA for SGLT1 or SGLT2 but did express the GLUT2, NHE-1 and NHE-3 proteins. Under control conditions, we observed a greater contribution of NHE-1 to pHi recovery relative to the other H(+) transporters. Acute high glucose treatment increased the HOE-694-sensitive pHi recovery rate and p-Erk1/2 and p90(RSK) abundance. These parameters were reduced by PD-98059, a Mek inhibitor (1 µM). Chronic high glucose treatment also increased the HOE-694-sensitive pHi recovery rate and p-p38MAPK abundance. Both parameters were reduced by SB-203580, a p38MAPK inhibitor (10 µM).

CONCLUSION

These results suggested that extracellular high glucose stimulated NHE-1 acutely and chronically through Mek/Erk1/2/p90(RSK) and p38MAPK pathways, respectively.