The discovery of marked elevations of circulating FGF23 levels in patients with CKD has challenged traditional theories regarding the origins of mineral metabolism defects in this setting. Given the complex interactions that exist between PTH, FGF23 and 1,25(OH)2D and a lack of longitudinal data evaluating these hormones at the onset and during the progression of renal failure, it has been difficult to discern the independent contributions of these hormones to the pathogenesis of CKD-MBD. With this investigation, we utilized the Col4a3 null mouse as a model of progressive kidney disease in order to gain new insight into the temporal relationship that exists between increments in FGF23, the development of mineral metabolism abnormalities, and the associated co-morbidities in CKD.
In our initial characterization of this model, Col4a3 null mice were found to develop progressive renal failure with substantial mortality by 12 weeks-of-age ( & ). More importantly, these mice demonstrate mineral metabolism abnormalities that recapitulate the changes reported in cross-sectional observations in humans with CKD-MBD (12
), including secondary hyperparathyroidism, a decline in circulating 1,25(OH)2
D levels, late hyperphosphatemia and hypocalcemia, and progressive elevations in FGF23 levels (). In addition, Col4a3 null mice develop late-stage high-turnover bone disease, characterized by osteoclast-mediated bone resorption (), that is consistent with the metabolic bone disease found in humans with elevated PTH levels in the setting of ESRD (14
). Interestingly, histological analysis also suggested a non-significant increase in bone volume in null mice, and no apparent defects in bone mineralization (i.e. no evidence of osteomalacia) or growth plate abnormalities. We found no consistent presence of vascular calcification in these mice (data not shown), possibly due to the short survival of these mice or the abbreviated duration of hyperphosphatemia. It is plausible that placing these mice on a diet containing higher levels of phosphate could promote a more reliable presence of vascular calcification by the 12 week timepoint.
Our next important observation in this model was evidence for an early rise in FGF23, prior to elevations in the traditional markers of renal function. To this end, increments of FGF23 in Col4a3 null mice were apparent by 6 weeks-of-age (), when parenchymal damage was first observed on renal histology () and a progressive rise in BUN and serum creatinine levels was not yet observed (). The significance of this observation could be substantial. Currently serum creatinine is the most commonly accepted marker for assessing a decline in glomerular function in patients with CKD, yet serum creatinine levels do not increase until a significant proportion of functional renal mass is lost (16
). The finding that FGF23 levels rise prior to creatinine levels suggests this hormone to have considerable potential as a predictor for kidney disease progression at an earlier stage of CKD. This seems consistent with cross-sectional data in humans suggesting an association of FGF23 with albuminuria in patients with known cardiovascular disease and only mild renal impairment (18
Our assessment of changes in serum parameters of mineral metabolism in Col4a3 null mice revealed a temporal association between elevations of FGF23 and reductions in serum 1,25(OH)2
D levels, consistent with the hypothesis that early elevations in serum FGF23 may contribute to decreased 1,25(OH)2
D levels in CKD by suppressing renal 1α-hydroxylase and stimulating 24-hydroxlase activity (19
). Similar to patients with CKD, this decline in serum 1,25(OH)2
D levels occurred despite a concomitant rise in PTH, which would be expected to stimulate 1,25(OH)2
D production by the kidney. This observation implies that the suppressive effects of FGF23 and phosphate retention may override the stimulatory effects of PTH on 1,25(OH)2
D production, at least in the setting of CKD. Unexpectedly, we observed unusually low PTH levels in Col4a3 null mice at 4 weeks-of-age compared to wild-type mice. It is possible that the low PTH levels may represent a transient suppression of PTH secretion by very early increments in FGF23, which would be consistent with the suspected direct action of FGF23 on the parathyroid gland (21
). Alternatively, the low PTH levels observed at this early time point may reflect some undefined interaction between early changes in glomerular physiology and pathways regulating PTH production. The recent finding that klotho and FGF-receptor 1 expression are decreased in parathyroid glands in advanced CKD may support the former hypothesis and explain the inability of FGF23 to suppress PTH production as kidney disease progresses in this model (22
). Furthermore, early decrements in parathyroid expression of klotho and FGF-receptor 1 may provide a mechanism for the rebound in PTH levels at 6 weeks-of-age in the face of unaltered serum calcium levels in the Col4a3 null model. Regardless, further studies are needed to determine the etiology and importance of these early changes in serum PTH levels, and to identify if these are transient changes that occur universally in the earliest stages of CKD, or a unique observation in Col4a3 null mice.
As renal function declined in Col4a3 null mice, we observed an increase in both the fractional excretion of phosphorus and total phosphorus excretion by 6 weeks-of-age (), that likely resulted from the increase in circulating levels of both PTH and FGF23. While previous studies in humans suggest that fractional excretion of phosphorus increases with advancing kidney disease (13
), an increase in total phosphorus excretion is not a common observation in this setting (13
), and may be a finding that is unique to either the Col4a3 null model or kidney diseases featuring a more rapid onset and progression. The observed increase in the fractional excretion of phosphorus at 6 weeks correlated with small decrements in sodium-phosphate co-transporter (NaPi-2a and NaPi-2c) and klotho gene expression (), with greater reductions occurring as renal failure progressed and PTH and FGF23 levels became exponentially elevated. While it has been previously speculated that a decline in renal klotho expression may be the inciting event for a rise in serum FGF23 levels in CKD, decrements in renal klotho expression in Col4a3 null mice occurred simultaneous to the initial rise in serum FGF23 levels, making it difficult to speculate which event occurred first in this model. Interestingly, serum phosphorus levels remained normal in Col4a3 null mice until the presence of severe kidney damage, when reduced renal klotho expression likely resulted in a resistance to the phosphaturic effects of FGF23 and a lack of residual functional nephron mass was present for the excretion of excess total body phosphate.
Similar to previous investigations in uremic animals (25
), our evaluation of Col4a3 null mice implicated bone as a source for elevated circulating FGF23 levels in advanced CKD, however, our data suggested that early increments in FGF23 may not result from increased production in bone (). This observation may help explain why circulating FGF23 levels are only modestly elevated in the early to middle stages of CKD (stages I–IV), yet become exponentially elevated in patients with end-stage disease (5
). It is plausible that the early rise in FGF23 in the setting of progressive kidney disease results from changes in excretion or post-transcriptional processing of this hormone. Alternatively, a tissue source other than bone may be contributing to the earliest elevations of circulating FGF23. Further studies will be needed to determine whether early increases in FGF23 in CKD are the result of extraskeletal tissue production or some post-transcriptional processing of this hormone. The pattern of FGF23 expression in bone from the Col4a3 null model proved to be unique from previous descriptions of FGF23 expression in inherited and genetic models of FGF23 over-expression (8
). In the hereditary hypophosphatemic models caused by mutations of Phex and Dmp1, the primary site of FGF23 production appears to be osteocytes in both cortical and trabelcular bone (8
). By contrast, Col4a3 null mice demonstrated little expression of FGF23 in cortical osteocytes, but widespread expression of FGF23 in trabecular osteocytes (). The observed difference in cell-type expression patterns for FGF23 production in bone between CKD and hereditary hypophosphatemic disorders may suggest an alternative mechanism for the stimulation of FGF23 production under these unique conditions.
We recognize that there are several limitations to this investigation. First, the relatively short time course for the progression to ESRD in Col4a3 null mice may have prevented the consistent development of several co-morbidities that are common in patients with ESRD, such as vascular calcification and osteomalacia. Second, it is plausible that different bone sites may have unique expression patterns of FGF23 in the setting of CKD, making it difficult to definitively preclude an increased production of FGF23 by bone in early CKD. Exhaustive analyses of FGF23 expression patterns in various skeletal locations will be needed to fully understand the contributions of bone to early increments of FGF23 in CKD. Third, despite an observed association between the rise in FGF23 and decline in 1,25(OH)2
D levels, we are unable to establish a causal relationship from these observations. Although a recent study investigating the effects of FGF23-deactivating antibodies on vitamin D metabolism suggests a role of FGF23 to promote 1,25(OH)2
D deficiency in this setting (28
). Finally, given the simultaneous rise of PTH and FGF23 that occurred in Col4a3 null mice, it is clear that an examination of time-dependent changes alone in this model will not be sufficient to decipher the complex interrelationship that exists between PTH and FGF23 in the setting of CKD.
Our data suggest FGF23 to be a novel biomarker for chronic kidney disease-mineral and bone disorder at the early stages of nephron loss. Additionally, our results imply that initial increments in circulating FGF23 may result from mechanisms other than increased FGF23 gene transcription in bone. While an early increase in FGF23 likely prevents hyperphosphatemia in the face of declining functional nephron mass, it appears that this occurs at the expense of promoting 1,25(OH)2D deficiency. While many controversies remain concerning the intricacies of mineral metabolism regulation in CKD-MBD and the best therapeutic approach to patients with this disorder, our successful characterization of the mineral metabolism phenotype of the Col4a3 null mouse suggests this model to possess considerable value to future hypothesis-driven research that will further advance our knowledge within this field.