Fig 1: cAMP signaling mediates lipocalin 2-induced bone stimulation of FGF23 production in osteoblasts. RNA sequencing of WT and Lcn2KO cortical bone identified 614 differentially regulated genes in Lcn2KO compared to WT (cutoffs: P < 0.05 and absolute fold change of 2). (a) Top 5 canonical pathways identified by ingenuity pathway analysis from the 614 genes dataset. (b) Normalized expression of dataset genes involved in the cAMP pathway with a twofold cutoff and P < 0.05 with the exception of *which indicates P < 0.1. (c) Fgf23 promoter activity in Control (Ctr) or Forskolin (FSK) -treated Fgf23 promoter-reporter MC3T3-E1 osteoblast cultures. (d) cAMP levels in Ctr, FSK, and LCN2 -treated MC3T3-E1 osteoblast cultures. (e) Representative micrograph and quantification (f) of western blotting detection of phosphorylated CREB (p-CREB) and total CREB, normalized to β-actin, in protein extracts from MC3T3-E1 osteoblasts treated with Ctr, FSK, and LCN2, and co-treated with LCN2 and cAMP inhibitor KT5720. Effects of Ctr, FSK, and LCN2 -treatment and KT5720 co-treatment on Fgf23 mRNA in MC3T3 osteoblasts (g) and promoter activity (h) in Fgf23 promoter-reporter MC3T3-E1 osteoblast cultures. Data are presented as mean ± SE, n ≥ 3 per group, P < 0.05 vs.* 6 h-Ctr, α 24 h-Ctr, and 6 h-FSK, £ 6 h-FSK + KT5720, β 6 h-LCN2. (i) Progressive alterations in kidney morphology and function induce inflammation-dependent lipocalin secretion leading to increased circulating LCN2. In bone, LCN2 increases FGF23 production through a cAMP/PKA/CREB-dependent mechanism, which contributes to excess FGF23 in CKD. Elevated FGF23 exerts pro-inflammatory effects, aggravating the inflammatory status in CKD. Excess FGF23 also targets the heart and contributes to the development of cardiac disease and mortality
Fig 2: Overexpression of lipocalin 2 in the brain induces astrocyte activation in the hippocampus. After a single intracerebroventricular injection of recombinant lipocalin 2 protein (7 μL, 1 μg/mL) or vehicle in the brain of the mice for 7 days, the brain coronal slices were fixed for immunohistochemistry with anti-GFAP antibody. Representative images were taken from the dorsal (A) and ventral (B) hippocampal CA3 areas, and the GFAP immunoreactivity was quantified as the percentage of GFAP-positive cells in the dorsal (C) and ventral (D) hippocampi of the vehicle and lipocalin 2 groups. Quantitative results are presented as mean ± SEM (n = 3 per group). The Student’s t-test was used to examine the significance of the mean. * p < 0.05 vs. the vehicle group. ** p < 0.01 vs. the vehicle group. GFAP: glial fibrillary acidic protein, LCN2: lipocalin 2, SEM: standard error of mean
Fig 3: Mice deficient in LCN2 expression have increased susceptibility to A. baumannii pneumonia.Lcn2-/- knockout mice and their WT C57BL/6 littermate controls were infected intranasally with WT A. baumannii. The infection was allowed to proceed for 36 h before the mice were humanely euthanized and the organs were harvested. Organs were homogenized, serially diluted in PBS and plated to LBA. The bacterial burdens of the kidneys (A), heart (B), liver (C), spleen (D), lungs (E), and blood (F) were determined. Each symbol represents the WT A. baumannii count in the corresponding organ of one animal. Data are compiled from two independent experiments. Statistical significance was determined by Mann-Whitney U test, where *p < 0.05, ** p < 0.01, and *** p < 0.001. The dashed line represents the limit of detection for the assay.
Fig 4: 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
Fig 5: Increased production of lipocalin 2 does not contribute to impaired kidney function in the Col4a3KO mouse model of chronic kidney disease. (a) Levels of serum lipocalin 2 (LCN2) measured in 4–23-week-old wild-type (WT) and Col4a3KO mice. (b) Levels of Lcn2 mRNA expression (reported to Rpl19 expression, and set at 1 in WT kidneys) in kidney, heart, bone, and bone marrow from 23-week-old WT and Col4a3KO mice. (c) Body weight, (d) blood urea nitrogen (BUN) levels, (e) 24 h urine albumin levels and (f) bright-field microscopy of hematoxylin & eosin (H&E) and picrosirius red (PSR, scale bar = 75 µm) stainings of kidneys from 23-week-old WT, Lcn2KO, Col4a3KO, and Col4a3KO/Lcn2KO (CPD) mice. P values were determined by 2-sided, paired t-test. Data are presented as mean ± SE, n ≥ 5 per group, P < 0.05 vs.*WT, $Lcn2KO
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