Lipotoxic disruption of NHE1 interaction with PI(4,5)P2 expedites proximal tubule apoptosis
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
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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Research Article Nephrology

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 7

LC-CoAs promote apoptosis through inhibition of NHE1 activity.

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LC-CoAs promote apoptosis through inhibition of NHE1 activity. (A) LLC-P...
(A) LLC-PK1 cells were incubated with glucose (25 mM, 24 hours), delipidated BSA (0.5%) complexed with palmitate (100 μM, 24 hours), etomoxir (100 μM, 24 hours), and triacsin C (5 μM, 12 hours) and assayed for apoptosis by TUNEL. Palmitate (400 μM) caused nearly 100% apoptosis, reflecting poor palmitoyl-CoA incorporation into lipid droplets (90) (n = 7 per group). *P < 0.05 compared to groups in lanes 6 or 7. (B) HRPT cells were stably transfected with ACSL1 or scrambled shRNAs or transiently transfected with adenoviral ACSL1 constructs and then assayed for apoptosis by TUNEL (n = 4–6 per group). *P < 0.05 compared to untreated cells. (C) LLC-PK1 cells were incubated with glucose (25 mM, 24 hours) with or without delipidated BSA (0.5%, 24 hours) complexed with palmitate or mixed NEFA (palmitate/stearate/oleate/linoleate = 3:1:4:2, 100 μM total concentration, 24 hours), etomoxir (100 μM, 24 hours), or triacsin C (5 μM, 12 hours), and then assayed by TUNEL (n = 4 per group). *P < 0.05 compared to other 2 groups. (D) LLC-PK1 cells were incubated with reagents as in A. NHE1 activity (dpHi/dT) was determined, as described in Methods (n = 3 per group). *P < 0.05 compared to untreated and triacsin C–treated groups. (E) HRPT cells were transfected as in B, and NHE1 activity was measured as in D (n = 4 per group). *P < 0.05 compared to untransfected cells. (F) C57BL/6 wild-type and Swe/Swe proximal tubule cells were incubated with reagents as in A. Apoptosis was measured by TUNEL (n = 3 per group). *P < 0.05 compared to untreated group.