Lipocalin 2 stimulates bone fibroblast growth factor 23 production in chronic kidney disease

Bone Research ARTICLE www.nature.com/boneres OPEN Lipocalin 2 stimulates bone fibroblast growth factor 23 production in chronic kidney disease 1234567890();,: Guillaume Courbon1, Connor Francis1, Claire Gerber1, Samantha Neuburg1, Xueyan Wang1, Emily Lynch1, Tamara Isakova1, Jodie L. Babitt2, Myles Wolf3, Aline Martin1 and Valentin David 1 Bone-produced fibroblast growth factor 23 (FGF23) increases in response to inflammation and iron deficiency and contributes to cardiovascular mortality in chronic kidney disease (CKD). Neutrophil gelatinase-associated lipocalin (NGAL or lipocalin 2; LCN2 the murine homolog) is a pro-inflammatory and iron-shuttling molecule that is secreted in response to kidney injury and may promote CKD progression. We investigated bone FGF23 regulation by circulating LCN2. At 23 weeks, Col4a3KO mice showed impaired kidney function, increased levels of kidney and serum LCN2, increased bone and serum FGF23, anemia, and left ventricular hypertrophy (LVH). Deletion of Lcn2 in CKD mice did not improve kidney function or anemia but prevented the development of LVH and improved survival in association with marked reductions in serum FGF23. Lcn2 deletion specifically prevented FGF23 elevations in response to inflammation, but not iron deficiency or phosphate, and administration of LCN2 increased serum FGF23 in healthy and CKD mice by stimulating Fgf23 transcription via activation of cAMP-mediated signaling in bone cells. These results show that kidney-produced LCN2 is an important mediator of increased FGF23 production by bone in response to inflammation and in CKD. LCN2 inhibition might represent a potential therapeutic approach to lower FGF23 and improve outcomes in CKD. Bone Research (2021)9:35 ; https://doi.org/10.1038/s41413-021-00154-0 INTRODUCTION Bone production of fibroblast growth factor 23 (FGF23) is increased in patients and animals with chronic kidney disease (CKD)1–3 and is associated with the development of left ventricular hypertrophy (LVH), heart failure, and mortality.1,2,4–7 Excess circulating FGF23 is the first major perturbation of mineral metabolism that occurs in CKD, however, the complex mechanisms that trigger elevations of FGF23 in CKD remain incompletely understood. Among these, multiple studies showed contributions of inflammation,8 iron deficiency,9 anemia,2 and local osteocyte defects.1 Notably, circulating FGF23 levels increase as kidney disease progresses, suggesting that kidney-bone crosstalk may contribute to excessive production of FGF23 by bone in response to kidney injury.10,11 Lipocalin 2, (LCN2) also known as neutrophil gelatinase-associated lipocalin in humans (NGAL) is a 25 kD lipophilic glycoprotein member of the lipocalin superfamily12 involved in innate immunity. The established role of LCN2 is to limit bacterial growth by binding to bacterial siderophores, which are low molecular weight chelators of ferric iron that are produced by bacteria to scavenge iron from their surrounding environment. In addition, LCN2 functions as an iron transporter by binding mammalian siderophores,13,14 and stabilizes labile iron/siderophore complexes.15,16 LCN2 allows cells to tolerate supra-physiological iron concentrations by scavenging free iron17–19 and protects against labile iron-mediated cytotoxicity. LCN2 is secreted by various cell types and tissues, including but not limited to immune cells,20 bone,21 liver,22 intestines,23 heart24 and kidney,25 and its expression is regulated mainly by infection and inflammatory status. In patients with acute and CKD, kidney production of NGAL/ LCN2 increases and can be detected in the urine and plasma; elevated urinary NGAL/LCN2 is a biomarker of acute kidney injury (AKI).25 Increased kidney LCN2 expression in AKI is thought to be a component of the systemic inflammatory response to AKI that helps redirect iron to support repair of renal tubular cells.26 In CKD, kidney expression and urine and serum LCN2 levels are also elevated, presumably in response to chronic kidney injury, inflammation, and infiltrating cells.27–30 Studies in which Lcn2 genetic deletion delayed CKD progression in mice demonstrate that LCN2 is not only a biomarker but could be a potential driver of CKD progression.31 Despite the links between LCN2 regulation and iron homeostasis, inflammation, and kidney disease, each of which is also involved in FGF23 regulation, potential direct relationships between LCN2, FGF23 regulation, and FGF23associated outcomes have not been studied. In the present study, we propose a novel mechanism to explain coincident increases in LCN2 and FGF23 soon after kidney injury,32 and the strong independent association between elevated levels of FGF23 and inflammatory markers.33 We hypothesized that bone is a target of kidney-secreted LCN2 and that increased LCN2 stimulates bone production of FGF23 in CKD. To test our hypothesis, we investigated the role of LCN2 in FGF23 regulation in health and in CKD. We show that circulating levels of LCN2 increased and paralleled CKD progression in the Col4a3KO mouse model of CKD, 1 Division of Nephrology and Hypertension, Department of Medicine, and Center for Translational Metabolism and Health, Institute for Public Health and Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA; 2Nephrology Division, Program in Membrane Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA and 3Division of Nephrology, Department of Medicine, and Duke Clinical Research Institute, Duke University School of Medicine, Durham, NC, USA Correspondence: Valentin David () Received: 10 September 2020 Revised: 23 March 2021 Accepted: 20 April 2021 © The Author(s) 2021 LCN2 and FGF23 in CKD G Courbon et al. 2 a b *$ 40 c *$ 500 000 *$ 500 000 25 20 15 10 -1 iFGF23/(pg·mL ) 50 000 * 30 cFGF23 (RU) NGAL/(ng·mL-1) 35 5 000 * 500 50 50 000 5 000 * 500 Healthy CKD stage 2-4 CKD stage 5 50 5 0 Healthy 2-4 5 5 Healthy 2-4 CKD stage 15 e R 2=0.79 Log iFGF23/(pg·mL-1) Log cFGF23 (RU) d 10 5 0 15 5 0 5 10 15 20 25 30 35 40 45 5 10 15 20 25 30 35 40 45 -1 g 15 2 R =0.54 Log iFGF23/(pg·mL-1) Log cFGF23 (RU) NGAL/(ng·mL ) R 2=0.57 10 5 0 5 CKD stage 10 -1 15 Healthy 2-4 R 2=0.73 NGAL/(ng·mL ) f 5 5 CKD stage 5 10 15 20 25 30 35 40 45 10 5 0 -1 NGAL/(ng·mL ) 5 10 15 20 25 30 35 40 45 NGAL/(ng·mL-1) Fig. 1 FGF23 and NGAL levels increase in patients with CKD. Levels of serum (a) NGAL, (b) total FGF23 (cFGF23), and (c) intact FGF23 (iFGF23) increase with the progression of kidney disease. NGAL correlates with both (d) and (f) cFGF23 (R2 = 0.79, partial correlation R2 = 0.57) and (e) and (g) iFGF23 (R2 = 0.73, partial correlation R2 = 0.54) levels, (d) and (e) unadjusted variables or (f) and (g) adjusted by eGFR. P values were determined by a two-sided, paired t-test. Values are mean ± SE, n ≥ 12/group, P < 0.05 vs. *Healthy, $Stage 2–4 and that kidney was the organ with the hi (...truncated)