Effects of atrial natriuretic peptide on mitochondria function in cortical collecting duct cells
Authors: Kashyrina Yuliia , Sultanova Regina , Nikiforova Anna , Ilatovskaya Daria 
Published: April 2020

Background

Atrial natriuretic peptide (ANP) is a hormone released from cardiomyocytes in response to cardiac wall stretching. ANP acts on the kidney by promoting sodium and water excretion as well as providing a general vasodilatory effect. Direct application of ANP has been shown to reduce sodium reabsorption in the renal tubular cells. Recently, it has been discovered that natriuretic peptides (NPs) are involved in lipolysis, lipid oxidation, and mitochondrial respiration. Evidence of a correlation between the activity of the NP system and mitochondrial respiration and biogenesis has been explored to some extent; however, little is known about these pathways in the kidney. Here we hypothesized that ANP may be implicated in mitochondrial uncoupling in the cortical collecting ducts (CCD), affecting sodium transport in this segment.

Methods

Mouse cortical collecting duct (mpkCCD) cells were cultured in DMEM/F12 medium with supplements and grown on glass‐bottom dishes, 96‐well plates or permeable supports. Cells were treated with ANP (5 nM to 10 μM range) for 1 or 24 hours. Fluorescent dyes TMRM and Rhod123, CellRox Deep Red, AmplexRed, Rhod2 and Hoechst 33342 were used to label mitochondrial membrane potential (TMRM/Rhod123), production of mitochondrial ROS, hydrogen peroxide, calcium, and nuclei in live cells, respectively. A Leica TCS SP5 confocal microscope or a BMG Labtech microplate reader were employed to monitor fluorescence intensity changes in response to ANP. Mitochondrial stress test assays were performed using a Seahorse XF96 Extracellular Flux Analyzer. Sodium currents in the polarized mpkCCD monolayers were measured with an EVOM multimeter. One‐way ANOVA (OriginPro) was used for statistical comparisons.

Results

Fluorescence microscopy with TMRM showed a mild (~30% at maximum effect) dose‐dependent decrease in mitochondrial membrane potential in mpkCCD cells treated with low dose ANP for 1, 2 and 24 h (1 nM to 500 nM), which was, interestingly, attenuated by higher ANP concentrations (>5 μM). These data were confirmed in a 96‐well plate assay using Rhod 123 (1 h and 24 hours). In addition, we observed an acute increase in ROS (and specifically, hydrogen peroxide) production and mitochondrial Ca2+ level in response to ANP (at 1 h), which is in line with the mild uncoupling reported above. Preliminary seahorse analysis indicated that ANP decreases mitochondrial respiration in these cells in a dose‐dependent manner. We have also shown that direct uncoupling of mitochondria with FCCP results in a dose‐dependent decrease in sodium currents mediated by Epithelial Sodium Channel (ENaC) as measured in short circuit current experiments.

Conclusion

Decreased mitochondrial membrane potential is indicative of a disruption in the oxidative phosphorylation, which is the main mechanism of ATP synthesis in the cell and the source of energy for Na+/K+‐ATPase (NKA). Since NKA provides the gradient for Na+ reabsorption in CCD, the reported mitochondria‐mediated effect suggests a novel pathway through which ANP may regulate Na+ reabsorption in the collecting ducts, potentially affecting blood pressure.

Support or Funding Information

APS Summer Undergraduate Fellowship Program; NIH R00 DK105160, Dialysis Clinic Inc Reserve Funds.