Fig 1: Model for the effects of phytate diets supplemented with low-Ca2+ or high-Ca2+ on Ca2+ and phosphate homeostasis.In the presence of a low-Ca2+ diet (Ca2+/phytate <5), phytate can adversely affect Ca2+ and phosphate homeostasis in normal healthy rats by inhibiting intestinal Ca2+ absorption while increasing phosphate overload through phytate hydrolysis in the intestine. These effects lead to crystal nephropathies, severe renal phosphate wasting, and pathologic bone loss via increased PTH and 1,25(OH)2D3. By contrast, in the presence of a high-Ca2+ diet, phytate forms multiple Ca2+-phytate salts, which makes them indigestible and unabsorbable in vivo, leading to their excretion in the feces as undigested Ca2+-phytate salts. This process prevents a vicious cycle of intestinal phosphate overloading and renal phosphate wasting while improving intestinal Ca2+ bioavailability, which eventually alleviates the detrimental effects of excess phytate hydrolysis.
Fig 2: Immunohistochemical staining of co-culture groups after 28 days of incubation. (A) Positive PTH staining was observed as brown cytoplasmic color in thyroid-parathyroid co-culture organoids in dif medium. (B) TTF1 staining was observed negative in thyroid-parathyroid co-culture organoids in dif medium. (C) TTF1 staining was observed positive with brown nuclear color (red arrows) in thyroid-parathyroid co-culture organoids in st medium. Scale bars represent (A) 20 µm and (B–C) 50 µm. dif = differentiation, PTH = parathormone, st = standard, TTF1 = thyroid transcription factor 1.
Fig 3: Immunofluorescence staining of proteins in parathyroid cells using confocal microscopy before differentiation protocol. (A) CaSR (membrane), (B) PTH (internal) and C) PTH1R (membrane), parathyroid cells stained in green for all proteins and counterstained in blue for nuclei. Objective 40 × original magnification, scale bars represent 20 µm. CaSR = calcium sensing receptor, PTH = parathormone, PTH1R= parathyroid hormone receptor 1.
Fig 4: High-phytate diets dysregulate mineral and phosphate metabolism in humans.(A) Experimental design (six healthy female volunteers). (B, C) Time course of serum levels of Ca2+ (B) and phosphate (C) for three different phytate doses. (D–H) Kinetics of urine pH (D), 24 hr urine Ca2+/Cr ratio (E), 24 hr urine Pi/Cr ratio (F), renal tubular Ca2+ reabsorption (TRCa) (G), and renal tubular phosphate reabsorption (TRP) (H) for three different phytate doses. (I) Time-course analysis of serum levels of intact PTH for different phytate doses. Data are presented as the mean ± SD for each dose (n = 6 per dose). Statistical significance was tested by repeated measure ANOVA between subjects and groups. All data are presented as the mean ± SD for each dose (n = 6 per dose).
Fig 5: Phytate-mediated Ca2+ deficiency promotes vitamin D insufficiency and renal phosphate wasting independent of FGF23 expression.(A–C) Time-course analysis of serum levels of intact PTH (A), 25(OH)D (B), and 1,25(OH)2D (C) in rats fed control, HP-LCa2+, and HP-HCa2+ diets. For the early measurement of 25(OH)D (B) and 1,25(OH)2D, we pooled the sera from 2 to 3 rats. (D–F) Time-course analysis of renal CYP27B1 (D), CYP24A1 (E), and VDR (F) in rats fed control, HP-LCa2+, and HP-HCa2+ diets. (G) Immunoblot analysis of renal αKlotho, NHERF1, NaPi-2a in rats fed control, HP-LCa2+, and HP-HCa2+ diets. (H) The levels of renal proteins were quantified using ImageJ software (NIH, Bethesda, MD, USA). (I) Immunoblot analysis of calvarial FGF23 in rats fed control, HP-LCa2+, and HP-HCa2+ diets. (J) The levels of total calvarial FGF23 were quantified using ImageJ software. All comparisons were conducted using two-way ANOVA with Tukey’s post hoc multiple comparison testing or the Kruskal–Wallis test (A–C) for non-parametric statistical analysis of data that did not have a normal distribution. Panels (A–C) failed the normality test, but no outliers were identified. All data are presented as the mean ± SD of each group (n = 4–6 per group). *, p<0.05; **, p<0.01; ***, p<0.001, compared with controls.
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