We used stored serum samples from 12 healthy volunteers and 36 patients with CKD stages 2–5 who participated in previous IRB-approved physiologic studies. All participants provided written informed consent to have their samples stored for future use for analysis of biomarkers related to kidney function and mineral metabolism. We measured NGAL using the NGAL ELISA assay (Abcam, Cambridge, MA, USA). We used a human intact FGF23 (iFGF23) enzyme-linked immunosorbent assay (ELISA) to measure the active iFGF23 protein and a C-terminal FGF23 (cFGF23) ELISA that recognizes the full-length protein and its C-terminal cleavage fragments to measure total FGF23 (both from Immutopics, Carlsbad, CA, USA).
Heterozygous C57Bl6/J-LCN+/−, expressing a neo-cassette targeting a 1.9 kb fragment in exons 2–4,39 obtained from Dr. Shizuo Akira at Osaka University were crossed to generate LCN2+/+ (WT) and LCN2−/− (Lcn2KO) mice. To assess the effects of acute inflammation, 12-week-old WT and Lcn2KO mice have injected ip with 250 ng·g−1 of recombinant murine interleukin 1 beta (IL-1β, Cell signaling). All diets were manufactured by Teklad (Envigo, Dever, CO, USA). Mice were maintained on a standard diet (Teklad 7012) except when otherwise specified. True iron deficiency was induced by feeding 3-week-old mice an iron deficient diet (TD.80396) or a mineral adjusted control diet (TD.80394) for 3 weeks. WT and Lcn2KO mice were fed a 2% high phosphate diet (TD.160039) or a mineral adjusted control diet (TD.80394) from 6 to 12 weeks of age. Six-week-old C57Bl6/J WT mice have injected ip during four days with murine recombinant LCN2 (50 ng·g−1 per day, 1857-LC-050, R&D systems, Minneapolis, MN, USA). Six-week-old C57Bl6/J WT mice were implanted ip with Alzet osmotic minipumps (Model 1004, Alzet, Cupertino, CA, USA) for 4 weeks to deliver murine recombinant LCN2 (5 or 50 ng·g−1 per day).
Heterozygous C57Bl6/J-Col4a3tm1Dec mice were crossed to LCN2+/−39 mice to generate C57Bl6/J- Col4a3+/+LCN2+/+ (WT), Col4a3+/+LCN2−/− (Lcn2KO), Col4a3−/−LCN2+/+ (Col4a3KO) and Col4a3−/−LCN2−/− (CPD). We harvested samples on a set of 23-week-old male littermates. We recorded body weight at sacrifice. In a separate set of animals, we recorded the age of death on Col4a3KO and CPD littermates to assess effects on lifespan. Col4a3KO and CPD mice have implanted subcutaneously with Alzet osmotic minipumps for 8 weeks to deliver 50 ng·g−1 per day of murine recombinant LCN2. All studies were approved by Institutional Animal Care and Use Committee at Northwestern University.
Biochemistry of mouse samples
We collected overnight urine samples from fasted animals housed overnight in metabolic cages and serum samples by intracardiac exsanguination. We used a murine intact FGF23 (iFGF23) enzyme-linked immunosorbent assay (ELISA) to measure the active iFGF23 protein and a C-terminal FGF23 (cFGF23) ELISA that recognizes the full-length protein and its C-terminal cleavage fragments to measure total FGF23 (both from Immutopics, Carlsbad, CA, USA). Phosphate, blood urea nitrogen (BUN), albumin, iron, and transferrin saturation were measured using colorimetric assays (Pointe Scientific, Canton, MI, USA). We measured serum ferritin using mouse ELISA assays (Alpco, Salem, NH, USA), circulating LCN2 using LCN2 mouse ELISA assay (Abcam, Cambridge, MA, USA), and erythropoietin using mouse EPO quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA).
RNA isolation, RT-PCR, and RNA sequencing
We isolated total RNA from tissues at sacrifice and from cell cultures using TRI reagent (Waltham, MA, USA) and purified RNA using RNeasy kit (Qiagen, Germantown, MD, USA).
For RNA sequencing, the total RNA library for each individual tibia was prepared using the TruSeq Total RNA-Seq Library Preparation Kit (Illumina, San Diego, CA), and the bar-coded cDNA libraries were sequenced for 75 bp single reads on one lane of Illumina NexSeq to generate a minimum of 100 million reads/library. Reads from each library were mapped to the mouse transcriptome and genome (UCSC mm10), filtered using StrandNGS software suite (Strand Life Sciences, Bangalore, Karnataka, India), and following Strand alignment and filtering pipelines. Reads were normalized using DESeq and we used baseline transformation to the median for each sample. FC and P value were calculated using moderated T-test and data were used for subsequent downstream pathway analyzes using the Ingenuity Pathway Analysis platform (IPA, Qiagen).
For RT-PCR, we synthesized first-strand cDNA (iScript cDNA Synthesis Kit, Bio-Rad Laboratories, Hercules, CA) and used the iCycler iQ real-time PCR detection system, iQ SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA), and adequate primer pairs for real-time quantitative PCR analysis. The threshold of detection of each gene expression was set at optimal reaction efficiency. The expression was plotted against a standard dilution curve of relative concentration, normalized to 60S ribosomal protein L19 (Rpl19) expression in the same sample, and expressed as fold change versus respective controls.
We performed echocardiography under isoflurane anesthesia 1 week prior to sacrifice (at 22 weeks of age) using a Vevo 770 High-Resolution Imaging System (VisualSonics, Toronto, ON, Canada). We used the parasternal short- and long-axis views to obtain two-dimensional and M-mode images as previously described.1,2,34
Hematologic parameters were acquired in whole blood using the HEMAVET 950 hematology system (Drew Scientific Inc., Oxford, CT, USA) and analyzed with multispecies software using mouse settings as previously described.2
Heart weight and tibia length were measured post sacrifice. Kidneys and hearts were collected at sacrifice, fixed in 100% ethanol, and embedded in paraffin. We collected 5-µm-thick sections using a rotary microtome. For analysis of the cardiac phenotype, we used cross-sections from the mid-chamber of the heart. We stained the sections with hematoxylin and eosin (H&E) to determine renal and cardiac morphology, picrosirius red (PSR) to determine kidney fibrosis. Images were acquired using light microscopy (Leica Microsystems, Buffalo Grove, IL, USA).
Cell cultures and assays
We cultured MC3T3-E1 osteoblastic cell lines (ATCC) according to American type culture collection guidelines. We prepared BMSCs from 6-week-old mice according to a previously described protocol.64 We maintained MC3T3-E1 and BMSCs in α-MEM containing 10% FBS, 10 U·mL–1 penicillin, and 100 μg·mL–1 streptomycin. For all experimental conditions, we plated MC3T3-E1 at 3 × 104 cells per well and BMSCs at 10 × 104 cells per well and cultured for 3 weeks in osteoblast-differentiating medium (α-minimal essential medium, 10% fetal bovine serum, 10 U·mL–1 penicillin, 100 μg·mL–1 streptomycin, 10 mmol·L–1 β-glycerophosphate, and 50 μg·mL–1 ascorbic acid; Sigma–Aldrich, St Louis, MO) prior to treatment and collection. To assess Fgf23 promoter activity, MC3T3-E1 cells were stably transfected with the pLuc-Fgf23 promoter plasmid carrying a secreted luciferase expression cassette under the control of the proximal Fgf23 promoter, a secreted alkaline phosphatase (SEALP) under the control of the CMV promoter, and a puromycin resistance cassette (Genecopoeia, Rockville, MD) as previously described.1,8 At day 21, cells were treated with recombinant murine 5–50 ng·mL–1 LCN2 (R&D systems), 10 μmol·L–1 FSK (Sigma Aldrich, St. Louis, MO, USA) and 12 μmol·L–1 KT5720 (Abcam). For promoter activity assays, Optimem medium containing 1% FBS 10 U•mL–1 penicillin and 100 μg·mL–1 streptomycin, supplemented with LCN2, FSK, and/or KT5720 was used for the last 24 h of culture. Promoter activity is represented by a relative luciferase unit normalized to pSEALP-CMV control. We conducted all experiments in triplicate.
Protein assays and SDS-Page
MC3T3/E1 osteoblast cells grown in the osteogenic medium were treated for 6 h with FSK, LCN2, or LCN2 + KT5720 in Opti-MEM with 1% FBS. Protein extracts were prepared using T-Per lysis buffer (Thermo Fisher Scientific, MA, USA) containing protease inhibitors cocktail and immunoblots were performed as previously described.2,8 For SDS-Page, 100 µg of total proteins were loaded on a 4-to-12% Bis-Tris (midi) Gel (Invitrogen). CREB expression was detected using rabbit antibodies against phosphorylated CREB (1:1 000; 9198) and CREB (1:1 000; 9197) (Cell Signaling Technology, MA, USA) and goat anti-β Actin (1:1 000, 8229) (Abcam, MA, USA). Cyclic AMP accumulation in cells was measured using a cAMP direct immunoassay (Abcam) from protein extracts in 100 µL of 0.1 mol·L–1 HCl.
Data are presented as mean ± SEM. Univariate and multiple regression, ANOVA followed by Fisher and two-sided, paired t tests were used for statistical inference using Statistica software (Statsoft, OK, USA). P values < 0.05 were considered statistically significant.
All human participants studies provided written informed consent to have their samples stored for future use for analysis of biomarkers related to kidney function and mineral metabolism and were enrolled in IRB-approved physiologic studies. All animal studies were approved by Institutional Animal Care and Use Committee at Northwestern University.