Epidermal growth factor activates Na(+/)H(+) exchanger in podocytes through a mechanism that involves Janus kinase and calmodulin

Abstract

Sodium-proton exchanger type 1 (NHE-1) is ubiquitously expressed, is activated by numerous growth factors, and plays significant roles in regulating intracellular pH and cellular volume, proliferation and cytoskeleton. Despite its importance, little is known about its regulation in renal glomerular podocytes. In the current work, we studied the regulation of NHE-1 activity by the epidermal growth factor receptor (EGFR) in cultured podocytes. RT-PCR demonstrated mRNAs for NHE-1 and NHE-2 in differentiated podocytes, as well as for EGFR subunits EGFR/ErbB1, Erb3, and ErbB4. EGF induced concentration-dependent increases in proton efflux in renal podocytes as assessed using a Cytosensor microphysiometer, were diminished in the presence of 5-(N-methyl-N-isobutyl) amiloride or in a sodium-free solution. Furthermore, pharmacological inhibitors of Janus kinase (Jak2) and calmodulin (CaM) attenuated EGF-induced NHE-1 activity. Co-immunoprecipitation studies determined that EGF induced formation of complexes between Jak2 and CaM, as well as between CaM and NHE-1. In addition, EGF increased levels of tyrosine phosphorylation of Jak2 and CaM. The EGFR kinase inhibitor, AG1478, blocked activation of NHE-1, but did not block EGF-induced phosphorylation of Jak2 or CaM. These results suggest that EGF induces NHE-1 activity in podocytes through two pathways: (1) EGF-->EGFR-->Jak2 activation (independent of EGFR tyrosine kinase activity)-->tyrosine phosphorylation of CaM-->CaM binding to NHE-1-->conformational change of NHE-1-->activation of NHE-1; and (2) EGF-->EGFR-->EGFR kinase activation-->association of CaM with NHE-1 (independent of Jak2)-->conformational change of NHE-1-->activation of NHE-1.

Figure 1. Immunofluorescence analysis of podocyte markers

Cells were fixed and then stained for synaptopodin or WT-1 to confirm differentiated from undifferentiated podocytes. Undifferentiated podocytes did not stain for synaptopodin (A). In contrast, differentiated podocytes stained for synaptopodin (B). Undifferentiated and differentiated podocytes stained for WT-1 (C and D).

Figure 2. Presence of functional EGFR in podocytes

A. RT-PCR analyses of EGFR/ErbB receptor subunit mRNA expression. Expression of different EGFR subunits in undifferentiated and differentiated podocytes is shown: EGFR (183 bp), Neu/HER2 (130 bp), ErbB3 (153 bp) and ErbB4 (204 bp). B. Microphysiometry was used to analyze the concentration-dependent effects of EGF on extracellular acidification rate (ECAR) in podocytes. Independent experiments were performed a minimum of three times in triplicate, and the data are presented as mean ± SEM of peak values.

Figure 3. EGF stimulates NHE-1 activity in podocytes

A. RT-PCR analysis of NHE isoforms. Expression of different NHE isoform mRNAs in (a) whole mouse kidney lysates; (b) undifferentiated podocytes; and in (c) differentiated podocytes are shown: NHE-1 (422 bp), NHE-2 (982 bp), NHE-3 (294 bp) and NHE-4 (993) bp. B. Microphysiometry was used to characterize EGF-induced increases in ECAR. Podocytes were stimulated with 10 ng/ml of EGF in the presence or absence of sodium-free medium or MIA (a NHE-1 inhibitor). Values depicted are mean ± SEM of peak values from 3 independent experiments performed in triplicate. **P < 0.01 vs drug alone samples.

Figure 4

Effects of CaM inhibitors and tyrosine kinase inhibitors on EGF-induced ECAR in podocytes. Microphysiometry was used to measure ECAR in podocytes. A. Podocytes were pretreated with CaM inhibitors for 30 min prior to stimulation with 10 ng/ ml of EGF. B. Podocytes were pretreated with AG1478 (20μM) or AG490 (50 μM) for 30 min prior to stimulation with 10 ng/ml of EGF. Values are mean ± SEM of peak values from 3 independent experiments performed in triplicate. * P < 0.05; **P < 0.01 vs drug alone samples.

Figure 5. EGF induces formation of signaling complexes between Jak2 and CaM, and NHE-1 and CaM in podocytes

Co-immunoprecipitation experiments were performed as described under Methods. A. Podocytes were pretreated with AG-490 (50 μM), or with AG-1478 (20 μM), or vehicle for 30 minutes, and then stimulated with 10 ng/ml of EGF for 5 min prior to being immunoprecipitated with agarose conjugated anti-Jak2 antibody and then immunoblotted with anti-CaM or anti-Jak2 antibody. The insert is a representative immunoblot (CaM). The same blot was stripped and re-probed with antibodies for total Jak2 to ensure a equal loading of proteins (Jak2). B. After stimulation of podocytes with 10 ng/ml of EGF for 10 min in the presence or absence of AG490 (50 μM) or AG1478 (20 μM), precleared cell lysates were immunoprecipitated with agarose conjugated anti-NHE-1 antibody and then immunoblotted with anti-calmodulin or anti-NHE-1 antibody. Values are mean ± SEM from 3 independent experiments. * P < 0.05 vs control, ^‡ P < 0.05 vs EGF.

Figure 5. EGF induces formation of signaling complexes between Jak2 and CaM, and NHE-1 and CaM in podocytes

Co-immunoprecipitation experiments were performed as described under Methods. A. Podocytes were pretreated with AG-490 (50 μM), or with AG-1478 (20 μM), or vehicle for 30 minutes, and then stimulated with 10 ng/ml of EGF for 5 min prior to being immunoprecipitated with agarose conjugated anti-Jak2 antibody and then immunoblotted with anti-CaM or anti-Jak2 antibody. The insert is a representative immunoblot (CaM). The same blot was stripped and re-probed with antibodies for total Jak2 to ensure a equal loading of proteins (Jak2). B. After stimulation of podocytes with 10 ng/ml of EGF for 10 min in the presence or absence of AG490 (50 μM) or AG1478 (20 μM), precleared cell lysates were immunoprecipitated with agarose conjugated anti-NHE-1 antibody and then immunoblotted with anti-calmodulin or anti-NHE-1 antibody. Values are mean ± SEM from 3 independent experiments. * P < 0.05 vs control, ^‡ P < 0.05 vs EGF.

Figure 6

Tyrosine phosphorylation of Jak2 and CaM in response to EGF treatment of podocytes. Co-immunoprecipitation experiments were performed as described under Methods. A. Podocytes were pretreated with AG-490 (50 μM), or with AG-1478 (20 μM), or vehicle for 30 minutes, and stimulated with 10 ng/ml of EGF for 5 min prior to cell lysates being immunoprecipitated with anti-phosphotyrosine/protein A-agarose, and then immunoblotted with polyclonal anti-EGFR antibody (A), or with polyclonal anti-Jak2 antibody (B). C. To determine if CaM was tyrosine phosphorylated, podocytes were stimulated with 10 ng/ml of EGF for 5 min after pretreatment with AG-490 (50 μM), or with AG-1478 (20 μM) or vehicle for 30 minutes, immunoprecipitated with monoclonal anti-phosphotyrosine (PY) antibody conjugated with protein A-agarose and then immunoblotted with a monoclonal anti-CaM antibody. The same blots were stripped and re-probed with polyclonal PY antibody to confirm that CaM is indeed tyrosine-phosphorylated in response to stimulation with EGF (not shown). Values are mean ± SEM from 5 independent experiments. * P < 0.05 vs control, ^‡ P < 0.05 vs EGF.

Figure 6

Tyrosine phosphorylation of Jak2 and CaM in response to EGF treatment of podocytes. Co-immunoprecipitation experiments were performed as described under Methods. A. Podocytes were pretreated with AG-490 (50 μM), or with AG-1478 (20 μM), or vehicle for 30 minutes, and stimulated with 10 ng/ml of EGF for 5 min prior to cell lysates being immunoprecipitated with anti-phosphotyrosine/protein A-agarose, and then immunoblotted with polyclonal anti-EGFR antibody (A), or with polyclonal anti-Jak2 antibody (B). C. To determine if CaM was tyrosine phosphorylated, podocytes were stimulated with 10 ng/ml of EGF for 5 min after pretreatment with AG-490 (50 μM), or with AG-1478 (20 μM) or vehicle for 30 minutes, immunoprecipitated with monoclonal anti-phosphotyrosine (PY) antibody conjugated with protein A-agarose and then immunoblotted with a monoclonal anti-CaM antibody. The same blots were stripped and re-probed with polyclonal PY antibody to confirm that CaM is indeed tyrosine-phosphorylated in response to stimulation with EGF (not shown). Values are mean ± SEM from 5 independent experiments. * P < 0.05 vs control, ^‡ P < 0.05 vs EGF.

Figure 7

Schematic representation of two converging pathways of activation of NHE-1 in podocytes by EGF. P-CaM indicates tyrosine-phosphorylated CaM. All other abbreviations are as described in the text.