Abstract
We have previously shown in renal cells that expression of the water channel Aquaporin-2 increases cell proliferation by a regulatory volume mechanism involving Na+/H+ exchanger isoform 2. Here, we investigated if Aquaporin-2 (AQP2) also modulates Na+/H+ exchanger isoform 1-dependent cell proliferation. We use two AQP2-expressing cortical collecting duct models: one constitutive (WT or AQP2-transfected RCCD1 cell line) and one inducible (control or vasopressin-induced mpkCCDc14 cell line). We found that Aquaporin-2 modifies Na+/H+ exchanger isoform 1 (NHE1) contribution to cell proliferation. In Aquaporin-2-expressing cells, Na+/H+ exchanger isoform 1 is anti-proliferative at physiological pH. In acid media, Na+/H+ exchanger isoform 1 contribution turned from anti-proliferative to proliferative only in AQP2-expressing cells. We also found that, in AQP2-expressing cells, NHE1-dependent proliferation changes parallel changes in stress fiber levels: at pH 7.4, Na+/H+ exchanger isoform 1 would favor stress fiber disassembly and, under acidosis, NHE1 would favor stress fiber assembly. Moreover, we found that Na+/H+ exchanger-dependent effects on proliferation linked to Aquaporin-2 relied on Transient Receptor Potential Subfamily V calcium channel activity. In conclusion, our data show that, in collecting duct cells, the water channel Aquaporin-2 modulates NHE1-dependent cell proliferation. In AQP2-expressing cells, at physiological pH, the Na+/H+ exchanger isoform 1 function is anti-proliferative and, at acidic pH, Na+/H+ exchanger isoform 1 function is proliferative. We propose that Na+/H+ exchanger isoform 1 modulates proliferation through an interplay with stress fiber formation.
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Acknowledgments
The authors thank Ricardo Dorr for his technical assistance.
Funding
This study was funded by grants from Fondo Nacional para la Ciencia y la Tecnología, Argentina [Grant Number: PICT 15-3525]; Universidad de Buenos Aires, Argentina [Grant Number: UBACYT 20020170100451BA and UBACYT 20020130100697BA]; and Consejo Nacional de Ciencia y Tecnología, Argentina [Grant Number: PIP 112 20130100057CO].
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Marina Mazzocchi and Gisela Di Giusto should be considered joint first authors: They have performed the majority of the experimental work. Micaela Porta performed some of the proliferation experiments. Alejandro Pizzoni has provided experimental design ideas and has worked in the microscope setup. Natalia Beltramone has participated in setting up the mpkCCDc14 cell model in nonpermeable supports. Paula Ford and Claudia Capurro have helped in the interpretation of the data and in revising the manuscript critically for important intellectual content. Valeria Rivarola has directed all the work providing the de majority of the design of the ideas and the experiments, the interpretation of the data, preparation of the manuscript and revising it critically for important intellectual content
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Marina Mazzocchi and Gisela Di Giusto should be considered joint first authors.
Key points
• NHE1 activity in AQP2-expressing cells is anti-proliferative at pH = 7.4 and proliferative at pH = 7.0.
• In the presence of AQP2, NHE1-dependent stress fiber formation modulates proliferation.
• Interaction between AQP2-TRPV calcium channels modulates NHE1-dependent proliferation.
Electronic supplementary material
Supplementary Fig. 1
AQP2 protein expression in mpkCDc14cells. Cells were exposed to vehicle (control) or 1 nM AVP (+AVP) for four days before experiments were performed. a: Representative images obtained after immunofluorescence studies using specific antibodies target to AQP2. b: Densitometric quantification of total membrane immunoblot bands, expressed as the AQP2 / β-Actin ratio. Bars are mean ± SEM from three independent experiments. ** p < 0.01, n = 3 when comparing control vs. AVP-stimulated mpkCCDc14 cells. Inset: Representative immunoblot using anti-AQP2 (28kD band) or β-Actin (42kD band) antibodies (PNG 621 kb)
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Mazzocchi, M., Di Giusto, G., Porta, M. et al. Na+/H+ exchanger isoform 1 activity in AQP2-expressing cells can be either proliferative or anti-proliferative depending on extracellular pH. J Physiol Biochem 76, 37–48 (2020). https://doi.org/10.1007/s13105-019-00713-4
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DOI: https://doi.org/10.1007/s13105-019-00713-4