Authors' Reply: Creatinine-Normalized Phosphaturia after Phosphate Loading in Skeletal Muscle–Specific Fgf23 Knockout Mice

We thank Drs. Yanmei Yin and Lixin Zhang for their thoughtful comments1 on our recent JASN article “Systemic Phosphate Elevations Induce fibroblast growth factor 23 (FGF23) Production in Skeletal Muscle to Reduce Renal Phosphate Reabsorption in Mice.”2 We appreciate their insightful discussion of our findings within the context of phosphate physiology.Figure 1: Amount of phosphate in 24-hour urine collections. We intraperitoneally injected FGF23 fl/ fl ; HSA- Cre + (KO) mice and their genetic controls (GC) with saline or Na2HPO4/NaH2PO4 solution (at 1 g phosphate [Pi] per kg body weight), and we kept the mice in metabolic cages for 24 hours. Total phosphate levels were analyzed in 24-hour urine collections (n=8–10; *P = 0.05; **P = 0.01; ***P = 0.001). Comparisons between groups were performed in a two-way ANOVA. FGF23, fibroblast growth factor 23; GC, genetic controls.We agree that in our phosphate injection study in mice with skeletal muscle–specific deletion of Fgf23 and in genetic control mice, the urine phosphate concentration does not necessarily have to be normalized to urine creatinine. Concentrations of substances in aliquots of urine are commonly normalized to the creatinine concentration to eliminate the confounding effect of urine volume. However, a 24-hour urine collection, as done here, eliminates the need for this step because the point of interest is the rate of phosphate excretion. Furthermore, the creatinine excretion rate is a function of muscle mass, which bears no obvious physiologic relationship to phosphate excretion. It is possible that the presented differences in phosphate excretion between Fgf23 knockout (KO) and control mice were blurred by normalization to creatinine excretion. However, we want to point out that the differences were nevertheless clear and significant. We also agree that it would be important to include creatinine-independent measures of renal phosphate excretion. We have analyzed total phosphate content in our 24-hour urine samples to determine total phosphate excretion (Figure 1). Compared with saline injection, phosphate injection elevated urine phosphate content from about 7 to 15 mg/24 h in control mice, while in Fgf23 KO mice, it only increased to about 9 mg/24 h. This difference in total urine phosphate content between phosphate-injected Fgf23 KO and control mice was statistically significant. We also want to point out that Fgf23 KO mice per se had no muscle phenotype and muscle mass was not different between Fgf23 KO and control mice, as shown in Figures 6B and 7B of the JASN article.2 Our molecular analyses of skeletal muscle tissue indicate that protein turnover was also not altered in Fgf23 KO mice (Figures 6, G–L, and 7, G–L). Combined with our finding that muscle function was not changed in Fgf23 KO mice (Figures 6A and 7A), these experiments suggest that skeletal muscle tissue does not undergo significant changes following Fgf23 deletion, and therefore, we would expect that creatinine excretion is also not different between Fgf23 KO and control mice. Combined, the findings presented in the JASN article2 and described here support our conclusion that skeletal muscle–derived FGF23 reduces renal phosphate reabsorption in mice.