High Glucose Concentration Stimulates NHE-1 Activity in Distal Nephron Cells: the Role of the Mek/Erk1/2/p90RSK and p38MAPK Signaling Pathways

Abstract

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 (pH_i) recovery was evaluated using microscopy in conjunction with the fluorescent probe BCECF/AM. The Na⁺-dependent pH_i 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 pH_i recovery relative to the other H⁺ transporters. Acute high glucose treatment increased the HOE-694-sensitive pH_i 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 pH_i 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.