Genetic Knockout and Rescue Studies in Mice Unravel Abnormal Phosphorus Threshold in Hypophosphatemic Rickets.

Maintaining phosphate homeostasis is of crucial biological importance for overall general health as inorganic phosphorus plays an important role in multiple biological processes, including skeletal mineralization, energy production, cell signaling, and regulation of protein function. Phosphate comprises ∼1% of total body weight, of which ∼85% is present in bone and teeth. The serum phosphate level is subject to regulation by dietary phosphate levels in addition to the gastrointestinal-bone-renal axis through a complex interplay between intestinal absorption, exchange with intracellular and bone storage pools, and renal tubular absorption (1). In the small intestine, phosphate is absorbed by paracellular transport, which depends on passive transfusion, and active transport, which occurs mainly through the sodium-dependent phosphate cotransporter sodium-dependent phosphate cotransporter (NPT)2b (2). In the kidney, most phosphate filtered through glomeruli is reabsorbed in proximal tubules via NPT2a and NPT2c (3). In bone, NPT2a, NPT2b, and PIT1 are known to be expressed in osteoblasts and could potentially be involved in transporting phosphate into the mineralizing front (3). In addition to the previously established roles of parathyroid hormone (PTH) and 1,25(OH)2D3 in the regulation of phosphate transport in the gastrointestinal-bone-renal axis, it is now becoming increasingly clear that fibroblast growth factor (FGF)23 plays a singular role in the dysregulation of phosphate homeostasis in various forms of hypophosphatemic rickets (4, 5).

Much of our understanding of mechanisms of phosphate regulation is derived from insights gained from human genetic disorders of phosphate homeostasis and genetically modified animal models exhibiting alterations in phosphate metabolism and mineralization abnormalities (4–6). Hypophosphatemic rickets is a heterogeneous disease caused by abnormalities in renal tubular reabsorption of phosphorus leading to phosphaturia, hypophosphatemia, poor bone mineralization, skeletal abnormalities, and occasionally fractures. Studies of the molecular basis for the heritable disorders of renal phosphate transport have led to the identification of gene abnormalities underlying X-linked hypophosphatemia (XLH) (Phex), autosomal dominant hypophosphatemic rickets (Fgf23), autosomal recessive hypophosphatemic rickets (ARHR)1 (Dmp1), ARHR2 (Enpp1), and ARHR3 (Fam20c) (7–11). Various genetic mouse models now reveal that although XLH, autosomal dominant hypophosphatemic rickets, and ARHR share a common pathogenesis mediated by changes in circulating FGF23 levels, the interplay between PTH, 1,25(OH)2D3, and FGF23 signaling pathways in regulating phosphate metabolism and skeletal phenotype in hypophosphatemic rickets is rather complex (12–14).

Activating mutation in the Phex gene, the most common cause of hypophosphatemic rickets, occurs in 1:20,000 people and is inherited as X-linked (7). The common abnormality reported in both X-linked and autosomal recessive hypophosphatemic rickets is an increase in circulating FGF23 due to enhanced skeletal expression as demonstrated by Ichikawa et al. (15) in the current issue of Endocrinology as well as by others. FGF23 interacts with the FGF receptor and Klotho to promote mitogen-activated protein kinase signaling and, thereby, decrease expression of Npt2a and Npt2c in renal tubular cells. Furthermore, activation of mitogen-activated protein kinase signaling also leads to decreased expression of Cyb27b1 and increased expression of Cyp24a1 resulting in reduced production of 1,25(OH)2D3 (16). These molecular changes together contribute to reduced reabsorption of phosphate in the kidney.

In a previous study, Econs and colleagues (17) demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, which further stimulates FGF23 production to maintain lower serum phosphate levels. In a study published in the current issue of Endocrinology (15), Econs and colleagues addressed the question of whether the set point for phosphate is also lowered in ARHR by using Dmp1 knockout mice, a murine model of ARHR. In the current study, the authors reported the presence of the same low extracellular phosphorus set point in Dmp1 null mice with the associated increase in both circulating and skeletal expression of Fgf23. These findings are compatible with clinical evidence that normalization of serum phosphorus is difficult to attain despite very high doses of phosphorus supplementation especially in growing children. The authors have further introduced Galnt3 null alleles to rescue the hypophosphatemia phenotype in the Dmp1 knockout mice. The Galnt3 gene encodes for polyptide N-acetylgalactosamine transferase 3, which has been shown to promote secretion of intact FGF23 by protecting FGF23 from proteolytic cleavage via O-glycosylation. Hyperphosphatemia, calcinosis, and an increased 1,25(OH)2D3 level are hallmarks of tumoral calcinosis in Galnt3 null mice (18). In the current study, Dmp1/;Galnt3/ mice showed severe hypophosphatemia at 4 weeks, but improved and, despite a higher FGF23, were closer to normal at 12 weeks of age both in terms of serum phosphate level and femur bone mineral density. These results suggest that Dmp1 knockout mice, like Phex mutant mice, have a lower set point for phosphorus.

Interestingly, this study found that the reduced serum phosphate in Dmp1 knockout mice seems unaffected by the disruption of the Galnt3 gene at 4 weeks of age, whereas serum phosphate was nearly normalized at 12 weeks of age. One potential explanation for why the serum phosphorus phenotype is normalized at 12 weeks but not at 4 weeks of age is that at 4 weeks of age the bones are rapidly forming and mineralizing and, therefore, exhibit higher demand for phosphorus at this age; the lack of functional GALNT3 may not be sufficient to meet the high phosphorus demand during this rapid growth period. The increase in serum phosphorus at 12 weeks in the double-knockout mice may be a reflection of decreased skeletal requirement in the older animals, a decline in renal phosphorus leak due to tubular maturity, or decreased responsiveness of the tubules to the circulating FGF23. Older children with hypophosphatemic rickets generally require less phosphorus supplementation as they grow older, an observation that mimics the findings of the current study.

Growth retardation is a common feature of XLH with greater shortness of limbs than trunk (19). Consistent with these clinical data, femur length is reduced considerably in Dmp1 knockout mice as well as in other genetic mouse models of hypophosphatemic rickets (8, 20). The reduced bone length in Dmp1 knockout mice is nearly rescued by deletion of Galnt4 alleles. These data, together with the finding that transgenic mice expressing FGF23 under the control of the α1(I) collagen promoter exhibited reduced femur length (12), suggest involvement of a FGF23-mediated mechanism in the retarded growth phenotype in hypophosphatemic rickets. Because FGF23 is mainly produced by osteocytes, the mechanisms by which endocrine FGF23 acts on growth plate chondrocytes to regulate longitudinal growth remain to be fully elucidated.

Another clinically relevant finding in the current study relates to the presence of hypocalcemia in the Dmp1 null animals, but not in double-knockout mice. Although not fully addressed by the authors, speculation about whether PTH or the lower 1,25(OH)2D3 played a role in the development of hypocalcemia would be interesting. There are no studies that have fully explained why hypocalcemia and secondary hyperparathyroidism develop in hypophosphatemic rickets. Future mechanistic studies utilizing the available genetic mouse models in the FGF23, PTH, and 1,25(OH)2D3 signaling pathways may help to resolve this issue.

In conclusion, the current study by Ichikawa et al. (15) has demonstrated that a lower set point for extracellular phosphorus in conjunction with elevated FGF23 may explain the hypophosphatemia and hypophosphatemic rickets seen in Dmp1 null mice. The issue of whether similar modulation of the phosphorus set point caused by altered FGF23 signaling is also a contributing factor in other forms of hypophosphatemic rickets requires future investigations.