ocytes are emerging as the focus of Tofacitinib 540737-29-9 intense investigation because they have been implicated as both targets and instigators of renal injury in various progressive renal diseases. Therefore, it would be desirable to elucidate signaling pathways in podocytes, which may be important in delineating the mechanisms through which podocytes contribute to the progression of glomerular injury. Because of the emerging role of the sodium proton exchanger type 1, also known as product of SLC9A1, solute carrier family 9A, type 1 in the regulation of the cytoskeleton, apoptosis and cellular proliferation, cell cycle control, and development and maintenance of the transformed cellular phenotype, we thought that it might be of interest to develop a better understanding of its regulation in podocytes.
Previous studies have demonstrated that EGF stimulates NHE 1 in non renal cells, but the signaling pathways involved in the regulation have not been fully elucidated. Moreover, currently there are no data on the regulation of NHE 1 in podocytes. In that regard, recent studies from our laboratory have shown that NHE 1 can be activated cyclooxygenase pathway by G protein coupled receptors or hypertonic medium, through Janus kinase 2 dependent phosphorylation of CaM, and subsequent interaction between CaM and NHE 1. Therefore, we wanted to determine whether EGF is important for regulating NHE 1 activity in podocytes, and to identify key components of the signal transduction pathway linking EGF and NHE 1. In the current study, we demonstrate that EGF stimulates NHE 1 activity in podocytes.
In addition, we have shown that Jak2 and CaM play critical roles in the activation of NHE 1 by EGF in podocytes. MATERIALS AND METHODS Cell Culture The podocyte cell line was kindly provided by Dr. Peter Mundel of Mt. Sinai School of Medicine. Podocytes were cultured as previously described. Undifferentiated podocytes were maintained in RPMI 1640 medium containing 10 units/ ml of mouse recombinant γ interferon, 10% FBS, 100 units/ml of penicillin and 100 g/ml of streptomycin at 33oC in 95% air and 5% CO2. To induce differentiation, podocytes were maintained in the same medium as undifferentiated podocytes without γ interferon at 37oC in 95% air and 5% CO2 for 14 days. All experiments were conducted using differentiated podocytes, unless stated otherwise. Immunofluorescence Microscopy Immunolabeling was performed as previously described.
Cells were seeded in 35 mm collagen coated glass bottom culture dishes and fixed with 2% paraformaldehyde, 4% sucrose in phosphate buffered saline for 10 min at room Coaxum et al. Page 2 Biochim Biophys Acta. Author manuscript, available in PMC 2012 May 31. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript temperature. Subsequently, cells were permeabilized with 0.3% Triton X 100 in PBS for 5 min, following which nonspecific binding sites were blocked with 2% fetal calf serum, 2% BSA and 0.2% gelatin in PBS for 1h. Incubations with the appropriate dilutions of primary and secondary antibodies were performed in blocking solution. The primary and secondary antibodies used were: anti WT1 , anti synaptopodin and Alexa Fluor 488 goat anti mouse IgG. Confocal microscopy was performed using a Zeiss LSM 510 META laser scanning microscope. Microphysiometry NHE 1 activity studies were conducted on a Cytosensor microphysiometer as previously described fo