Phosphate salt selection affects mortality and vascular calcification in the adenine-induced chronic kidney disease mouse model

Abstract

Patients with chronic kidney disease (CKD) are at an increased risk for vascular calcification (VC), a strong predictor of cardiovascular events. Rodent models with elevated dietary levels of adenine and phosphate are commonly used to study CKD-induced VC; however, dietary composition, regimen duration, and reported outcomes vary widely across studies. Researchers customize high-adenine, high-phosphate (AdeHP) diets by adjusting phosphate levels and selecting specific phosphate salts as the phosphate source. In this observational study, we found that the selection and amount of phosphate salt used as the dietary phosphate source in an AdeHP mouse model of CKD-induced VC influence disease severity and survival in a sex-specific manner. Mice were fed a high-adenine diet for 6 wk to induce CKD, followed by an AdeHP diet containing either sodium (AdeHP-HNa) or calcium (AdeHP-HCa) based phosphate salts for up to six additional weeks to induce VC. Mice on the AdeHP-HCa diet exhibited worse kidney function and higher circulating phosphate levels compared with those on the AdeHP-HNa diet, with males showing more severe outcomes than females. However, mice on the AdeHP-HCa diet showed lower VC levels compared with those on the AdeHP-HNa diet, with males developing more calcification than females after 6 wk of the AdeHP regimen. Most importantly, a pronounced sex-dependent difference in survival was observed in males on the AdeHP-HNa diet, with only 16.67% surviving. These findings underscore the importance of phosphate salt selection and sex-specific variability in modeling CKD-induced VC with the AdeHP diet, providing key insights and considerations for preclinical studies.NEW & NOTEWORTHY We identified that the type of phosphate salt used to increase dietary phosphate in the adenine-induced CKD mouse model of VC influences calcification severity, survival, renal function, and circulating phosphate levels in a sex- and time-specific manner, with males exhibiting more severe outcomes. These findings highlight the importance of selecting and reporting phosphate salts used to supplement custom-made diets to prevent animal mortality and effectively model CKD-induced VC.

Keywords: adenine diet; chronic kidney disease; phosphate salts; sex differences; vascular calcification.

Figure 1.. Experimental design, body weight trajectories, and survival analysis of mice on phosphate-modified diets to induce CKD and vascular calcification.

A) Schematic of the experimental design. Adult (8–10 weeks old) C57BL/6J male (M ■) and female (F ○) mice were assigned to either a standard chow diet (control) or a high-adenine diet for 6 weeks, followed by a high-adenine, high-phosphate diet with either sodium (AdeHP-HNa) or calcium (AdeHP-HCa) phosphate for up to 6 additional weeks. Body weight was recorded every 3 weeks; serum and aortas were collected at weeks A9HP3 and A12HP6 for further analysis. B–C) Body weight over the 12-week period in B) male and C) female mice on standard chow, AdeHP-HNa, and AdeHP-HCa diets. Data are presented as mean ± SD. Statistical significance was assessed using two-way ANOVA with Tukey’s post hoc test comparing chow to AdeHP+HNa (*) and chow to AdeHP+HCa (#) throughout regimen duration for each sex (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001). Sample sizes at each timepoint are detailed in Supplementary Table 1. D–E) Kaplan-Meier survival curves comparing male (––) and female (––––) mice over the 12-week diet regimen for D) AdeHP-HNa (male n = 42, female n = 21) and E) AdeHP-HCa (male n = 28, female n = 24). At the end of the regimen, 7/42 males and 17/21 females survived on the AdeHP-HNa, and 27/28 males and 24/24 females on the AdeHP-HCa, respectively. The AdeHP-HNa diet significantly (p < 0.0001) reduced survival rate in mice compared to AdeHP-HCa diet. Survival differences were evaluated using log-rank (Mantel-Cox) tests.

Figure 2.. Phosphate levels, kidney function, and vascular calcification in mice on high-adenine, high-phosphate diets differ by duration, phosphate source, and sex.

A–B) Serum phosphate concentrations in male (M ■) and female (F ○) mice after at weeks A) A9HP3 and B) A12HP6 on the high-adenine, high-phosphate diets. Both AdeHP-HNa and AdeHP-HCa diets significantly elevated phosphate compared to standard chow. C–D) Circulating blood urea nitrogen (BUN) levels as an indicator of kidney function at C) A9HP3 and D) A12HP6 timepoints. BUN levels worsened with longer duration on diet and were influenced by phosphate salt source and sex. E–H) Quantification of aortic calcification intensity with corresponding mean intensity imaging at F,H) A9HP3 and E,G) A12HP6 timepoints. Mice on the AdeHP-HNa diet showed greater calcification than those on AdeHP-HCa, with increases observed over time and between sexes. Each point represents an individual animal. Statistical significance was assessed by ANOVA with Tukey’s post hoc test. Sample sizes per group are listed in Supplementary Tables 2–4.