Lipotoxic disruption of NHE1 interaction with PI(4,5)P2 expedites proximal tubule apoptosis
Shenaz Khan, … , Eckhard Ficker, Jeffrey R. Schelling
Shenaz Khan, … , Eckhard Ficker, Jeffrey R. Schelling
Published February 17, 2014
Citation Information: J Clin Invest. 2014;124(3):1057-1068. https://doi.org/10.1172/JCI71863.
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Lipotoxic disruption of NHE1 interaction with PI(4,5)P2 expedites proximal tubule apoptosis

Abstract

Chronic kidney disease progression can be predicted based on the degree of tubular atrophy, which is the result of proximal tubule apoptosis. The Na+/H+ exchanger NHE1 regulates proximal tubule cell survival through interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], but pathophysiologic triggers for NHE1 inactivation are unknown. Because glomerular injury permits proximal tubule luminal exposure and reabsorption of fatty acid/albumin complexes, we hypothesized that accumulation of amphipathic, long-chain acyl-CoA (LC-CoA) metabolites stimulates lipoapoptosis by competing with the structurally similar PI(4,5)P2 for NHE1 binding. Kidneys from mouse models of progressive, albuminuric kidney disease exhibited increased fatty acids, LC-CoAs, and caspase-2–dependent proximal tubule lipoapoptosis. LC-CoAs and the cytosolic domain of NHE1 directly interacted, with an affinity comparable to that of the PI(4,5)P2-NHE1 interaction, and competing LC-CoAs disrupted binding of the NHE1 cytosolic tail to PI(4,5)P2. Inhibition of LC-CoA catabolism reduced NHE1 activity and enhanced apoptosis, whereas inhibition of proximal tubule LC-CoA generation preserved NHE1 activity and protected against apoptosis. Our data indicate that albuminuria/lipiduria enhances lipotoxin delivery to the proximal tubule and accumulation of LC-CoAs contributes to tubular atrophy by severing the NHE1-PI(4,5)P2 interaction, thereby lowering the apoptotic threshold. Furthermore, these data suggest that NHE1 functions as a metabolic sensor for lipotoxicity.

Authors

Shenaz Khan, Bassam G. Abu Jawdeh, Monu Goel, William P. Schilling, Mark D. Parker, Michelle A. Puchowicz, Satya P. Yadav, Raymond C. Harris, Ashraf El-Meanawy, Malcolm Hoshi, Krekwit Shinlapawittayatorn, Isabelle Deschênes, Eckhard Ficker, Jeffrey R. Schelling

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Figure 5

LC-CoAs compete with PI(4,5)P2 for NHE1 binding.

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LC-CoAs compete with PI(4,5)P2 for NHE1 binding. (A–C) Swe/Swe proximal ...
(A–C) Swe/Swe proximal tubule cells were transfected with and without NHE1-RFP acceptor and incubated with glucose (25 mM, 24 hours), delipidated 0.5% BSA plus palmitate (100 μM, 24 hours), and/or etomoxir (100 μM, 24 hours) to enhance intracellular LC-CoA concentration. FRET was measured using the relative fluorescence intensity of the donor [BODIPY-PI(4,5)P2, excitation/ emission λ = 488/510] from TIRF images (original magnification, ×60), according to Methods. (A) Epifluorescence image of transfected and untransfected cells. (B) TIRF image of BODIPY fluorescence for corresponding cells in A. (C) Energy transfer efficiency between PI(4,5)P2-BODIPY and NHE1-RFP by TIRF microscopy using the formula E = 1 – FDA/FD, where E stands for energy transfer efficiency, FDA is the fluorescence intensity of the donor BODIPY-PI(4,5)P2 in cells expressing the acceptor RFP-NHE1, and FD is the fluorescence intensity of the donor BODIPY-PI(4,5)P2 in the absence of the acceptor RFP-NHE1 (n = 20 cells per condition). *P < 0.05 compared to other group. (D–I) Xenopus oocytes microinjected with (D and F–I) cRNA encoding EGFP-tagged cNHE1 (22.5 ng in 25 μl) or (E) H2O were pretreated for 24 hours at 37°C with (F) albumin (0.5%); (G) albumin and palmitate (100 μM); (H) albumin, palmitate, and etomoxir (100 μM); and (I) albumin, palmitate, and triacsin C (5 μM). Cells were fixed in 4% paraformaldehyde and viewed by confocal microscopy. Original magnification, ×20. (J and K) cRNA encoding EGFP-tagged cNHE1 (22.5 ng in 25 μl) and increasing concentrations (estimated 50 μM–500 μM final concentration) of (J) palmitoyl-CoA or (K) malonyl-CoA, viewed by confocal microscopy. Original magnification, ×40.