Over 26 million people in the U.S. have chronic kidney diseases, most commonly from diabetic glomerular injury. However, progression to end stage is more tightly coupled to proximal tubule disappearance (tubular atrophy), which is caused by apoptosis. The NHE1 Na+/H+ exchanger inhibits renal tubular epithelial cell (RTC) apoptosis by binding to the membrane phosphoinositide PI(4,5)P2. Intracellular accumulation of non-esterified fatty acid (NEFA) metabolites stimulates apoptosis. Multiple factors converge to cause RTC NEFA accumulation in diabetic nephropathy (DN), including reabsorption of filtered albumin bound to NEFA. Following transport into cells NEFA are esterified to form amphipathic long-chain acyl-Coenzyme A (LC-CoA) intermediates, which are structurally similar to PI(4,5)P2, and preferentially used as an energy source. Excess LC-CoAs are stored as cytoplasmic triglyceride to shield cells from lipotoxicity, though NEFA buffering capacity is limited in RTC. Preliminary data demonstrate that NEFA, LC-CoA and triglyceride concentrations are elevated in RTC from mouse models of DN. In vitro, PI(4,5)P2 and LC-CoAs bound NHE1 with similar affinities;PI(4,5)P2 stimulated, and LC-CoAs inhibited NHE1-dependent Na+/H+ exchange and cell survival. We hypothesize that in DN, NEFA bound to albumin are reabsorbed by proximal tubules. The formations of LC- CoAs, which are poorly metabolized, accumulate in RTC and lead to cytotoxity. Surplus LC-CoAs compete with PI(4,5)P2 for binding to the NHE1 cytosolic tail, which leads to failure of NHE1-dependent Na+/H+ exchange and RTC apoptosis, resulting in tubular atrophy and progressive kidney disease. To characterize NEFA and LC-CoA regulation of RTC dysfunction, control and diabetic nephropathy (eNOS-/- db/db) mice will be assayed for kidney NEFA and LC-CoA concentration, and 13C-labeled NEFA uptake. The impact of rate-limiting, NEFA-metabolizing enzymes acyl CoA synthetase, acyl CoA thioesterase and carnitine palmitoyl transferase will be addressed using pharmacologic inhibitors and knockout mice. All mice will be phenotyped by histochemical staining for triacylglycerol, apoptosis, and interstitial fibrosis;for renal function by serum creatinine and uine albumin: creatinine ratios;for ex vivo NHE1 activity by fluorescence methods. In vitro, pharmacologic and genetic gain and loss of function approaches will be applied to alter LC- CoA concentration in wild-type and NHE1-null RTC, which will be analyzed for apoptosis and NHE1 activity. To determine whether LC-CoAs compete with PI(4,5)P2 for binding and regulation of NHE1, competition of LC- CoA for PI(4,5)P2 binding to NHE1 will be determined in vitro using fluorescence microscopy techniques in cells, phospholipids overlays and displacement of fluorescently labeled PI(4,5)P2 bound to NHE1 immobilized on Sepharose beads. NHE1 activity will be assessed in intact cells and whole cells patches following manipulation of LC-CoAs and PI(4,5)P2 content.

Public Health Relevance

Over 500,000 people in the U.S. have kidney failure that requires dialysis or transplantation to stay alive. The purpose of this project is to determine why kidneys fail. In particular, we plan to study biochemical pathways that prevent kidneys cells from dying, which may lead to new therapies to halt the progression of kidney diseases.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (KMBD)
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Ketchum, Christian J
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Case Western Reserve University
Internal Medicine/Medicine
Schools of Medicine
United States
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Khan, Shenaz; Cabral, Pablo D; Schilling, William P et al. (2018) Kidney Proximal Tubule Lipoapoptosis Is Regulated by Fatty Acid Transporter-2 (FATP2). J Am Soc Nephrol 29:81-91
Schelling, Jeffrey R (2016) Tubular atrophy in the pathogenesis of chronic kidney disease progression. Pediatr Nephrol 31:693-706
Parker, Mark D; Myers, Evan J; Schelling, Jeffrey R (2015) Na+-H+ exchanger-1 (NHE1) regulation in kidney proximal tubule. Cell Mol Life Sci 72:2061-74
Lakhe-Reddy, Sujata; Li, Vincent; Arnold, Thomas D et al. (2014) Mesangial cell ?v?8-integrin regulates glomerular capillary integrity and repair. Am J Physiol Renal Physiol 306:F1400-9
Khan, Shenaz; Abu Jawdeh, Bassam G; Goel, Monu et al. (2014) Lipotoxic disruption of NHE1 interaction with PI(4,5)P2 expedites proximal tubule apoptosis. J Clin Invest 124:1057-68
Abu Jawdeh, Bassam G; Khan, Shenaz; DeschĂȘnes, Isabelle et al. (2011) Phosphoinositide binding differentially regulates NHE1 Na+/H+ exchanger-dependent proximal tubule cell survival. J Biol Chem 286:42435-45
Khan, Shenaz; Lakhe-Reddy, Sujata; McCarty, Joseph H et al. (2011) Mesangial cell integrin ?v?8 provides glomerular endothelial cell cytoprotection by sequestering TGF-? and regulating PECAM-1. Am J Pathol 178:609-20
Kim, Jane H; Konieczkowski, Martha; Mukherjee, Amitava et al. (2010) Podocyte injury induces nuclear translocation of WTIP via microtubule-dependent transport. J Biol Chem 285:9995-10004
Holthouser, Kristine A; Mandal, Amritlal; Merchant, Michael L et al. (2010) Ouabain stimulates Na-K-ATPase through a sodium/hydrogen exchanger-1 (NHE-1)-dependent mechanism in human kidney proximal tubule cells. Am J Physiol Renal Physiol 299:F77-90
Abu Jawdeh, Bassam G; Kanso, Abbas A; Schelling, Jeffrey R (2009) Evidence-based approach for prevention of radiocontrast-induced nephropathy. J Hosp Med 4:500-6

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