This study examined the effects of chronic, 6-week FGF23-neutralizing Ab treatment on secondary HPT and comorbidities in a rat CKD model. Many of the mineral disturbances and comorbidities observed in humans with CKD-MBD are also prevalent in this model, including low levels of serum vitamin D and calcium with increased serum phosphate load and cardiovascular complications, such as LVH. Serum FGF23 levels also rise in 5/6Nx HPD rats, as is observed in humans with CKD (32
The dosing frequency and FGF23-neutralizing activity of the rat anti-rat FGF23-Ab utilized in this study was determined in vivo by demonstrating a rapid and sustained rise in serum phosphate in normal mice and rats following a single dose of FGF23-Ab. These results were consistent with earlier findings with a different FGF23-Ab (31
In the current study, FGF23-Ab treatment led to higher serum vitamin D, calcium, and phosphate and lower serum PTH levels for up to 5 weeks compared with those of CKD control rats. Changes in FEPi
were variable, and 24-hour urinary phosphate was not affected by FGF23-Ab treatment. Thus, the treatment-induced worsening of hyperphosphatemia may not be explained solely by lower FEPi
. The FGF23-Ab–induced elevations in vitamin D, which increases intestinal phosphate uptake, may have partially contributed to hyperphosphatemia in these animals. The serum PTH reductions observed up to week 5 in the high-dose group may have reflected the PTH-lowering effect of increased circulating vitamin D and calcium. Alternatively, FGF23 may have directly regulated PTH expression and secretion by the parathyroid gland (33
). Unfortunately, the FGF23-induced suppression of secondary HPT by FGF23-Ab treatment was not sustained in the high-dose group beyond 5 weeks of treatment, when an upward trend in serum PTH levels and an associated serum phosphate load increase were observed in some rats in the high-dose group. Specifically, at week 6, 4 rats had serum PTH levels (range 3870 to 13 409 pg/ml) 2-fold higher than baseline and serum phosphate levels (range 17.3 to 38.0 mg/dl) 3- to 4-fold higher than baseline.
Most importantly, more ME rats were found in the high-dose group than in the low-dose and control Ab 5/6Nx groups. The increased mortality in the FGF23-Ab high-dose group was at least partially attributed to renal failure and was not due to Ab load, since the 5/6Nx control Ab group received an equivalent amount (10 mg/kg) of control Ab with the same dosing frequency.
CKD rats in this study were fed a HPD, which raises the question of whether certain results, such as the observed increase in vascular calcification in the FGF23-Ab–treated animals, might have been different using a normal- or low-phosphate–containing diet. The HPD was chosen because it simulates the Western diet of many CKD patients (34
) and potentiates disease progression in the 5/6Nx rats, which facilitates studying this disease model. It is possible that a normal- or low-phosphate diet would lessen the extent of FGF23-Ab–induced mineral imbalances, thus preventing the occurrence of vascular calcifications observed in this study. However, the observed elevation in serum phosphate is a predicted pharmacodynamic outcome of FGF23 neutralization when functional target tissue (i.e., kidney) is present. Indeed, in other studies in normal mice and CKD rats fed a normal-phosphate–containing diet, FGF23-neutralizing Abs also increased serum phosphate, but the long-term effect of FGF23 neutralization on vascular calcification was not examined (31
). The appearance of soft tissue calcifications is also observed in FGF23-null mice that exhibit deregulated mineral metabolism (3
). Future studies using dietary or pharmacological control of phosphate and studies using phosphate and FGF23 clamping are needed to better determine the direct contribution of impaired phosphate and mineral homeostasis to the pronounced vascular calcification and morbidity observed upon FGF23-Ab treatment in this study.
The increased serum calcium caused by FGF23-Ab treatment likely resulted from the higher serum vitamin D, which would lead to increased intestinal calcium absorption. Concordantly, FGF23-null mice exhibited elevated serum vitamin D and calcium despite lower serum PTH (3
). The increased urinary calcium observed in FGF23-Ab–treated CKD rats is consistent with higher serum calcium and lower tubular calcium reabsorption in response to reduced serum PTH levels brought about by prolonged suppression of FGF23, higher vitamin D and calcium, and contrasts with findings in FGF23-overexpressing mice (39
). The contribution of increased osteoclast activity to higher serum calcium (and phosphate) levels after treatment is unknown, but remains a formal possibility given the increase in osteoclast surface observed at the end of the study in FGF23-Ab–treated CKD rats.
The lack of an FGF23-Ab effect on renal 1
expression likely reflects the opposing actions of elevated circulating PTH and FGF23 at week 6. Additionally, lack of FGF23-Ab’s effect on NaP2a
expression is consistent with no observed effect on FEPi
at week 6 and likely also due to the higher serum levels of PTH as a negative regulator of NaP2a (40
), at this final time point.
The observation in this study that FGF23-Ab treatment deregulated mineral homeostasis and caused vascular calcification coupled with published data from FGF23 loss-of-function mutations in mice that also exhibit elevated serum phosphate and increased soft tissue and vessel calcifications in the presence of a functioning kidney (3
) implicate a contribution of increased serum phosphate levels to this pathology. FGF23-Ab–treated rats with aortic calcifications had normal or elevated serum calcium and higher serum phosphate than that found in 5/6Nx control Ab rats, whose aortae did not calcify. Although unlikely, due to the continued elevations in serum phosphate during the treatment period that indicated FGF23 activity was continuously inhibited, we cannot exclude the possibility that the high-dose FGF23-Ab elicited a compensatory response in FGF23 that may have had a direct impact on the vessel wall. Given data implicating elevated serum phosphate, potentiated by increased serum vitamin D, in promoting vascular calcification (42
), sustained elevations of high serum phosphate in the presence of elevated vitamin D likely contributed directly to the vascular calcification observed in the high-dose group.
Numerous studies have correlated increased serum FGF23 levels with CKD disease progression, LVH, cardiovascular disease, and mortality (22
). Recently, local and systemic FGF23 administration to normal mice induced pathological heart remodeling and marker gene expression (27
). Together with in vitro studies using cardiomyocytes, these studies indicated that FGF23 can have a direct impact on the heart. In our study, heart/body weights were higher for 5/6Nx rats, and expression of pathological heart hypertrophic markers were elevated, despite minimal LVH. Significant LVH may have required a longer time to manifest and or more detailed quantitative histomorphometric microanalyses. Nonetheless, we detected no treatment effect of FGF23-Ab on the cardiac parameters measured. Our study differed from that by Faul et al. (27
), who administered a nonspecific FGF receptor inhibitor to 5/6Nx rats fed a normal diet immediately after 5/6Nx surgery for 14 days, whereas FGF23-Ab was dosed after established renal failure (6 weeks after 5/6Nx) in HPD-fed rats for an additional 6 weeks. As mentioned, our findings are complicated by disturbances in mineral homeostasis that resulted from longer-term FGF23-Ab treatment, whereas no such disturbances were reported by Faul et al. (27
It is of interest that even in the context of deregulated mineral homeostasis, FGF23-Ab treatment improved the hyperparathyroid bone disease observed in CKD-MBD 5/6Nx rats. The observed improvements in bone parameters were consistent with the degree of serum PTH reduction resulting from FGF23-Ab administration. The described relationship between improved bone mineralization and decreased vascular calcifications (44
) was not observed in this model. It is likely that the contrasting effects of FGF23-Ab on bone and the vasculature were, in part, secondary to effects on vitamin D, phosphate, and PTH. Increased serum vitamin D and decreased serum PTH likely contributed to improvements in bone turnover and mineralization observed in this model, whereas elevated serum vitamin D and concomitant disturbances in mineral balance caused by FGF23 neutralization promoted calcification.
In conclusion, in a rat model of early CKD fed a HPD, chronic neutralization of FGF23 increased serum vitamin D, decreased secondary HPT, and improved numerous bone parameters. Unfortunately, these beneficial effects were countered by increased serum phosphate levels, resulting in part from the inhibition of FGF23’s phosphaturic activity on the residual kidney, which likely contributed to the increased mortality and vascular calcification observed in rats treated with the high dose of FGF23-Ab. Additional studies are needed to assess the effect of FGF23 neutralization in CKD-MBD rats with adequately controlled phosphate levels and at different stages of CKD, including end-stage renal disease. In dialysis patients, FGF23 levels are significantly elevated, and the phosphaturic effect of FGF23 is less relevant because of diminished renal function. Whether neutralizing FGF23 in these individuals would ameliorate the proposed deleterious extrarenal effects of supraphysiological FGF23 on the cardiovascular system (27
) while obviating the impairments of mineral homeostasis described in this manuscript warrants further studies in preclinical models.