Phosphate Restriction Prevents Metabolic Acidosis and Curbs Rise in FGF23 and Mortality in Murine Folic Acid-Induced AKI.

SIGNIFICANCE STATEMENT: Patients with AKI suffer a staggering mortality rate of approximately 30%. Fibroblast growth factor 23 (FGF23) and phosphate (P i ) rise rapidly after the onset of AKI and have both been independently associated with ensuing morbidity and mortality. This study demonstrates that dietary P i restriction markedly diminished the early rise in plasma FGF23 and prevented the rise in plasma P i , parathyroid hormone, and calcitriol in mice with folic acid-induced AKI (FA-AKI). Furthermore, the study provides evidence for P i -sensitive osseous Fgf23 mRNA expression and reveals that P i restriction mitigated calciprotein particles (CPPs) formation, inflammation, acidosis, cardiac electrical disturbances, and mortality in mice with FA-AKI. These findings suggest that P i restriction may have a prophylactic potential in patients at risk for AKI. BACKGROUND: In AKI, plasma FGF23 and P i rise rapidly and are independently associated with disease severity and outcome. METHODS: The effects of normal (NP) and low (LP) dietary P i were investigated in mice with FA-AKI after 3, 24, and 48 hours and 14 days. RESULTS: After 24 hours of AKI, the LP diet curbed the rise in plasma FGF23 and prevented that of parathyroid hormone and calcitriol as well as of osseous but not splenic or thymic Fgf23 mRNA expression. The absence of Pth prevented the rise in calcitriol and reduced the elevation of FGF23 in FA-AKI with the NP diet. Furthermore, the LP diet attenuated the rise in renal and plasma IL-6 and mitigated the decline in renal α -Klotho. After 48 hours, the LP diet further dampened renal IL-6 expression and resulted in lower urinary neutrophil gelatinase-associated lipocalin. In addition, the LP diet prevented the increased formation of CPPs. Fourteen days after AKI induction, the LP diet group maintained less elevated plasma FGF23 levels and had greater survival than the NP diet group. This was associated with prevention of metabolic acidosis, hypocalcemia, hyperkalemia, and cardiac electrical disturbances. CONCLUSIONS: This study reveals P i -sensitive FGF23 expression in the bone but not in the thymus or spleen in FA-AKI and demonstrates that P i restriction mitigates CPP formation, inflammation, acidosis, and mortality in this model. These results suggest that dietary P i restriction could have prophylactic potential in patients at risk for AKI.

A novel method for automated crystal visualization and quantification in murine folic acid-induced acute kidney injury.

Folic acid (FA)-induced acute kidney injury (FA-AKI) is an increasingly prevalent rodent disease model involving the injection of a high dose of FA that culminates in renal FA crystal deposition and injury. However, the literature characterizing the FA-AKI model is sparse and dated in part due to the absence of a well-described methodology for the visualization and quantification of renal FA crystals. Using widely available materials and tools, we developed a straightforward and crystal-preserving histological protocol that can be coupled with automated imaging for renal FA crystal visualization and generated an automated macro for downstream crystal content quantification. The applicability of the method was demonstrated by characterizing the model in male and female C57BL6/JRj mice after 3 and 30 h of FA treatment. Kidneys from both sexes and timepoints showed a bimodal distribution of FA crystal deposition in the cortical and medullary regions while, compared with males, females exhibited higher renal FA crystal content at the 30-h timepoint accompanied by greater kidney weight and higher plasma urea. Despite comparable plasma phosphate concentrations, FA-AKI resulted in a substantially more elevated plasma intact fibroblast growth factor 23 (FGF23) in females, reflected by a similar pattern in osseous Fgf23 mRNA expression. Therefore, the presented method constitutes a valuable tool for the quantification of renal FA crystals, which can aid the mechanistic characterization of the FA-AKI model and serves as a means to control for confounding changes in FA crystallization when using the model for investigating early and prophylactic AKI therapeutic interventions.NEW & NOTEWORTHY Here, we describe a novel method for the visualization and quantification of renal folic acid (FA) crystals in the rodent FA-induced acute kidney injury (FA-AKI) model. The protocol involves a straightforward histological approach followed by fully automated imaging and quantification steps. Applicability was confirmed by showing that the FA-AKI model is sex-dependent. The method can serve as a tool to aid in characterizing FA-AKI and to control for studies investigating prophylactic therapeutic avenues using FA-AKI.

Intact FGF23 predicts serum phosphate improvement after combined nicotinamide and phosphate binder treatment in hemodialysis patients.

Background: Hyperphosphatemia is associated with increased mortality and cardiovascular morbidity of end-stage kidney failure (ESKF) patients. Managing serum phosphate in ESKF patients is challenging and mostly based on limiting intestinal phosphate absorption with low phosphate diets and phosphate binders (PB). In a multi-centric, double-blinded, placebo-controlled study cohort of maintenance hemodialysis patients with hyperphosphatemia, we demonstrated the efficacy of nicotinamide modified release (NAMR) formulation treatment in addition to standard PB therapy in decreasing serum phosphate. Here we aimed to assess the relationship between phosphate, FGF23, inflammation and iron metabolism in this cohort. Methods: We measured the plasma concentrations of intact fibroblast growth factor 23 (iFGF23) and selected proinflammatory cytokines at baseline and Week 12 after initiating treatment. Results: We observed a strong correlation between iFGF23 and cFGF23 (C-terminal fragment plus iFGF23). We identified iFGF23 as a better predictor of changes in serum phosphate induced by NAMR and PB treatment compared with cFGF23. Recursive partitioning revealed at baseline and Week 12, that iFGF23 and cFGF23 together with T50 propensity were the most important predictors of serum phosphate, whereas intact parathyroid hormone (iPTH) played a minor role in this model. Furthermore, we found serum phosphate and iPTH as the best predictors of iFGF23 and cFGF23. Sex, age, body mass index, and markers of inflammation and iron metabolism had only a minor impact in predicting FGF23. Conclusion: Lowering serum phosphate in ESKF patients may depend highly on iFGF23 which is correlated to cFGF23 levels. Serum phosphate was the most important predictor of plasma FGF23 in this ESKF cohort.

Blocking FGF23 signaling improves the growth plate of mice with X-linked hypophosphatemia.

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone. X-linked hypophosphatemia (XLH) is the most prevalent inherited phosphate wasting disorder due to mutations in the PHEX gene, which cause elevated circulating FGF23 levels. Clinically, it is characterized by growth impairment and defective mineralization of bones and teeth. Treatment of XLH is challenging. Since 2018, neutralizing antibodies against FGF23 have dramatically improved the therapy of XLH patients, although not all patients fully respond to the treatment, and it is very costly. C-terminal fragments of FGF23 have recently emerged as blockers of intact FGF23 signaling. Here, we analyzed the effect on growth and bone of a short 26 residues long C-terminal FGF23 (cFGF23) fragment and two N-acetylated and C-amidated cFGF23 peptides using young XLH mice (Phex C733RMhda mice). Although no major changes in blood parameters were observed after 7 days of treatment with these peptides, bone length and growth plate structure improved. The modified peptides accelerated the growth rate probably by improving growth plate structure and dynamics. The processes of chondrocyte proliferation, death, hypertrophy, and the cartilaginous composition in the growth plate were partially improved in young treated XLH mice. In conclusion, these findings contribute to understand the role of FGF23 signaling in growth plate metabolism and show that this may occur despite continuous hypophosphatemia.

Oxidatively-mediated in silico epimerization of a highly amyloidogenic segment in the human calcitonin hormone (hCT15-19).

In order to study the effects of peptide exposure to oxidative attack, we chose a model reaction in which the hydroxyl radical discretely abstracts a hydrogen atom from the α-carbon of each residue of a highly amyloidogenic region in the human calcitonin hormone, hCT15-19. Based on a combined Molecular Mechanics / Quantum Mechanics approach, the extended and folded L- and D-configuration and radical intermediate hCT15-19 peptides were optimized to obtain their compactness, secondary structure and relative thermodynamic data. The results suggest that the epimerization of residues is generally an exergonic process that can explain the cumulative nature of molecular aging. Moreover, the configurational inversion induced conformational changes can cause protein dysfunction. The epimerization of the central residue to the D-configuration induced a hairpin structure in hCT15-19, concomitant with a possible oligomerization of human calcitonin into Aß(1-42)-like amyloid fibrils present in patients suffering from Alzheimer's disease.