Journal: Kidney international

Article Title: Soluble α-klotho and heparin modulate the pathologic cardiac actions of fibroblast growth factor 23 in chronic kidney disease.

doi: 10.1016/j.kint.2022.03.028

Figure Lengend Snippet: Figure 2 | Production and detection of bioactive soluble klotho (sKL), a fibroblast growth factor (FGF) 23 binding protein with antihypertrophic effects. (a) Neonatal rat ventricular myocytes were cotreated with FGF23 (25 ng/ml) or vehicle (phosphate-buffered saline [PBS]) and with sKL (100 ng/ml), anti-FGF23 blocking antibody (1 mg/ml), or vehicle. FGF23 treatments caused an increase in cell area, while sKL and anti-FGF23 blocked this effect. (b,e) Standard curves (linear regression) for the measurements of sKL using our plate-based assay, with FGF23 coated on 96-well plates (Thermo Fisher Scientific) and incubation with Fc-FGFR1c, followed by horseradish peroxidase (HRP)– coupled anti-Fc antibody and HRP for detection. sKL was applied in (b) assay buffer or (e) in rat serum as a diluent. For sKL in buffer, the assay gives a linear range allowing the calculation of sKL concentrations in samples with a limit of detection (LOD) of w300 pg/ml. For sKL in serum, the assay is w10 less sensitive, with an LOD of w10 ng/ml. Absorbances measured are depicted in arbitrary units (AUs) as individual dots and standard curves as straight lines. (c) Two commonly used commercial preparations of sKL (Com.sKL) and of the first extracellular domain of klotho (KL1) were analyzed in our sKL detection assay in comparison to our own sKL. Our sKL protein showed w7 higher binding activity than did Com.sKL, and we detected no binding of KL1. (d) Using our FGF23/Fc-FGFR1c–based detection assay, we analyzed binding affinities of different variants and isoforms of klotho, that is, sKL and full-length klotho (KL), as well as soluble b-klotho (sbKL) and full-length b-klotho (bKL). Human embryonic kidney 293T (HEK293T) cells were stably transfected with klotho constructs or a green fluorescent protein (GFP) vector used as a negative control, and total cell lysates were analyzed. A strong signal was observed for sKL and much weaker binding for KL. GFP, sbKL, and bKL showed no measurable binding activity. (f) Rats were injected via the tail vein with sKL (100 mg/kg) or vehicle (PBS), and serial bleeds were taken at different time points and analyzed by our FGF23/Fc-FGFR1c–based detection assay. At the 15-minute time point, high levels of sKL were detected in sKL-injected rats compared with vehicle-injected controls. sKL exhibited a half-life of <15 minutes, and starting at 3 hours postinjection, we could no longer detect sKL. (g) Rats were injected with sKL or vehicle as before, but serial bleeds were taken at earlier time points. After w10 minutes postinjection, sKL levels were reduced by about half. Comparison between groups was performed using 1-way analysis of variance followed by a post hoc Tukey test (a) or using 2-tailed t tests (f,g). A significance level of P # 0.05 was accepted as statistically significant. (a) n ¼ 3 independent isolations, 50 cells per slide, 150 total cells per condition. *P # 0.05 versus all other groups. (c,d) n ¼ 3. (f,g) n ¼ 3. *P # 0.05 versus vehicle treated at the same time point, #P # 0.05 versus same treatment in the preceding time point. For plate-based assays, n ¼ 3 replicate wells.

Article Snippet: Secondary antibodies are horseradish peroxidase (HRP)–conjugated anti-goat (V8051, Promega), anti-mouse (W4021, Promega), and anti-rabbit (W4011, Promega) for Western blotting; HRP-coupled anti-human antibody (109035098, Jackson ImmunoResearch Laboratories) and anti-FLAG (A8592, SigmaAldrich) for the plate-based binding assay; and Cy3-conjugated goat–anti-mouse (115165166, Jackson ImmunoResearch Laboratories) for immunocytochemistry.

Techniques: Binding Assay, Saline, Blocking Assay, Incubation, Detection Assay, Comparison, Activity Assay, Stable Transfection, Transfection, Construct, Plasmid Preparation, Negative Control, Injection