Partial Answers from Partial Klotho Deficiency
Authors: Tamara Isakova, Myles Wolf
Journal: Journal of the American Society of Nephrology (2012)

Membrane-bound Klotho is the coreceptor for fibroblast growth factor 23 (FGF23) that increases its affinity for FGF receptors (FGFRs).1 Binding of FGF23 to FGFR-Klotho complexes in the kidney stimulates phosphaturia and inhibits production of 1,25-dihydroxyvitamin D.2 In the parathyroid glands, FGF23-FGFR-Klotho signaling also inhibits parathyroid hormone (PTH) secretion.3 Alternative splicing or cleavage of the extracellular domain of Klotho yields a soluble form of the protein, which possesses phosphaturic effects and also activates calcium reabsorption by transient receptor potential vanilloid 5 in the distal nephron.4 Landmark studies of genetically altered mice helped illuminate the interdependent roles of Klotho and FGF23 in calcium and phosphate homeostasis. Klotho-ablated (kl/kl) mice develop severe hyperphosphatemia, markedly elevated levels of FGF23, endogenous 1,25-dihydroxyvitamin D intoxication with resultant hypercalcemia, suppressed levels of PTH, and hypercalciuria.4–6 The animals also manifest accelerated aging that can be prevented by a low phosphate diet or ablation of CYP27B1, which ameliorate the biochemical alterations.7,8 These data suggest that disordered mineral metabolism contributes directly to shortened lifespan of kl/kl mice. Recent reports of direct adverse effects of Klotho deficiency on the vasculature and FGF23 excess on the myocardium suggest that these factors may also play a contributory pathophysiological role in the premature death associated with Klotho deficiency.9,10 The study by Olauson et al. in this issue of JASN11 sheds new light on this rapidly evolving field. The investigators genetically engineered mice with partial Klotho deletion limited to the distal tubule (Ksp-Kl−/−), which is the predominant site of renal Klotho expression, in order to define the contribution of kidney-specific Klotho deficiency to the abnormalities observed in published reports of kl/kl. As expected, the variable degree of renal Klotho deletion introduced much more phenotypic variability than in complete knockout models but also allowed an evaluation of dose-response relationships between Klotho expression levels and biochemical phenotype. Unlike kl/kl, Ksp/Kl−/− mice demonstrate a normal gross phenotype, preserved fertility, no vascular calcification, and normal renal parenchyma devoid of the intrarenal calcifications and interstitial fibrosis that develop in kl/kl mice. From a biochemical perspective, partial Klotho deletion is associated with hyperphosphatemia, elevated FGF23 levels, and reduced PTH, but serum calcium and 1,25-dihydroxyvitamin D levels were not significantly different compared with wild-type animals. Many of these results recapitulate previously reported findings from kl/kl, albeit in milder form, which is consistent with the partial knockout. However, critical distinctions between partial and complete Klotho ablation reveal additional important nuances of the feedback loops that regulate mineral metabolism and their potential effects on the cardiovascular system. One critical difference is that PTH is similarly suppressed in Ksp-Kl−/− as in kl/kl mice, but in the Ksp-Kl−/− this occurred in the absence of hypercalcemia or elevated 1,25-dihydroxyvitamin D levels, which are thought to be the main mechanisms for PTH suppression in kl/kl mice. One explanation for this finding the authors propose is that secondary increases in FGF23 suppressed PTH directly by activating intact FGFR-Klotho complexes in the parathyroid glands.3 Although convincing, we must be cautious not to dismiss a critical role of altered calcium homeostasis based on the animals’ normal serum calcium and 1,25-dihydroxyvitamin D levels. Rather than using mass spectroscopy, serum 1,25-dihydroxyvitamin D levels were measured by RIA, which is far less precise, and thus, limited statistical power may have obscured biologically relevant increases in 1,25-dihydroxyvitamin D levels. This is most strikingly demonstrated in Table 1 of the manuscript in which 1,25-dihydroxyvitamin D levels were nearly two-fold (11) higher in Ksp-Kl−/− than wild-type but the results were not statistically significant. In the same table, the most significant finding was markedly increased renal expression of CYP27B1. This suggests that the expected increase in 1,25-dihydroxyvitamin D levels may actually be present in the model but escaped detection, as the authors speculate. Furthermore, high-normal 1,25-dihydroxyvitamin D levels in the setting of hyperphosphatemia and low PTH should likely be interpreted as inappropriately high just as low-normal 1,25-dihydroxyvitamin D levels are inappropriately low in hypophosphatemic disorders of primary FGF23 excess such as X-linked hypophosphatemia. Inappropriately elevated 1,25-dihydroxyvitamin D levels can suppress PTH directly through activation of the vitamin D receptor, but enhanced gut absorption of calcium is another critical mechanism of vitamin D–mediated PTH suppression that might be operative in the current model despite the lack of hypercalcemia. It is interesting to postulate that low levels of PTH in the setting of preserved kidney structure and function in Ksp-Kl−/− versus kl/kl might have promoted an adequately high urinary calcium clearance to avoid hypercalcemia; although serum creatinine was only modestly higher in kl/kl, the animals are much smaller and have reduced muscle mass. This interpretation would imply a tendency toward positive calcium balance as the driver of reduced PTH. However, one could also invoke negative calcium balance driven by hypercalciuria that is caused by a primary defect in Klotho-mediated calcium reabsorption in the distal tubule.4 Unfortunately, this study did not report on the bone phenotype of Ksp-Kl−/− mice, which could have shed light on this question by providing greater insight into the animals’ calcium balance. The finding that dietary phosphate loading raised PTH levels in Ksp-Kl−/− mice despite further increases in FGF23 is another interesting finding. This mirrors the clinical observation of secondary hyperparathyroidism that complicates oral phosphate repletion therapy in X-linked hypophosphatemia. Unfortunately, serum and urinary calcium levels of animals exposed to the high phosphate diet are not reported. If these were significantly lower than on a lower phosphate diet, as might be expected, one could hypothesize that PTH escaped from FGF23 inhibition in response to the threat of calcium depletion. Alternatively, perhaps FGF23 is simply a less important regulator of parathyroid function in vivo than currently considered. Parathyroid-specific Klotho deletion might be particularly helpful in addressing this issue. Another critical difference between Ksp-Kl−/− and kl/kl mice that is of high relevance to nephrologists and investigators in the field is the absence of vascular calcification in Ksp-Kl−/− despite hyperphosphatemia and elevated FGF23 levels. Previous studies that manipulated dietary phosphate intake and vitamin D metabolism to prolong survival of kl/kl mice suggest that the biochemical abnormalities of mineral metabolism cause lethal complications such as vascular calcification.7,8 Perhaps the Ksp-Kl−/− mice did not calcify because hyperphosphatemia was less severe, because of lack of hypercalcemia, or because of vascular protection by persistent vascular Klotho expression.9 We are not told whether the Ksp-Kl−/− mice develop calcification on a high phosphate diet when serum phosphate levels went up significantly and FGF23 skyrocketed. If there still were no calcification in this setting, it would raise interesting new questions regarding the role of phosphate as a mediator of calcification, such as whether vascular Klotho deficiency is an obligate cofactor for hyperphosphatemia-mediated calcification in vivo. As a corollary, perhaps variable expression of soluble or vascular Klotho could represent a novel mechanism to explain why some chronically hyperphosphatemic patients do not develop calcification. Finally, lack of calcification in the setting of severely elevated FGF23 levels implies no direct effect of FGF23 despite suggestive epidemiologic data.12 The work by Olauson et al. provides important new data, but there are two important questions for which this study could only provide partial answers. The first regards our poor understanding of the regulation of FGF23 production. Interesting new insights emerge from analyses in which the investigators compared wild-type and Ksp-Kl−/− animals that were matched for serum phosphate. The finding that FGF23 levels were significantly elevated in Ksp-Kl−/− animals despite identical serum phosphate levels is consistent with prior data that did not show a direct effect of serum phosphate on FGF23 regulation.13 Furthermore, the investigators demonstrated an inversely linear relationship between serum phosphate and renal Klotho expression, whereas for FGF23 there was a strong threshold effect when Klotho expression was reduced to <30% of wild-type. This dissociation of the relationships between serum phosphate and FGF23 levels relative to Klotho expression also suggests that serum phosphate may not be the primary regulator of FGF23 production. Perhaps increased 1,25-dihydroxyvitamin D levels or increased calcium absorption is the major determinant of FGF23 production in Klotho deficiency. Indeed, recent data implicate calcium loading as a novel stimulus for FGF23,14 and CYP27B1 ablation in Klotho null mice leads to FGF23 deficiency.7 Further investigation in the area is clearly needed. The second issue that remains elusive is whether disordered mineral metabolism in human CKD results from FGF23 resistance due to a primary deficiency of Klotho or if a primary rise in FGF23 promotes secondary downregulation of Klotho expression. This study provides clear evidence that FGF23 levels increase in response to reduced Klotho expression. Previous work from the same group using mice engineered to overexpress FGF23 demonstrates suppression of Klotho expression.15 There is, therefore, no doubt about the reciprocal interrelationship between Klotho expression and FGF23 levels. However, the authors’ suggestion that elevated FGF23 in the Ksp-Kl−/− mice indicates that human CKD may be conceptualized as a state of FGF23 resistance is premature. Although the intact renal function and partial Klotho deficiency in the Ksp-Kl−/− makes it more like early CKD than the kl/kl, several aspects of the Ksp-Kl−/− mice reported by Olauson et al. are entirely inconsistent with human CKD, which is characterized by decreased calciuria, increased fractional excretion of phosphate, elevated FGF23 and PTH in the setting of normal serum phosphate, and reduced 1,25-dihydroxyvitamin D levels. Thus, the quest to crystallize the complex pathophysiological cascade of disordered mineral metabolism in human CKD continues. Disclosures T.I. received honoraria from Genzyme and Shire. M.W. has served as a consultant or received honoraria from Abbott, Amgen, Diasorin, Genzyme, Kai, Luitpold, Mitsubishi, and Shire.