C-Terminal Fibroblast Growth Factor 23, Iron ...

CLINICAL EPIDEMIOLOGY

www.jasn.org

C-Terminal Fibroblast Growth Factor 23, Iron Deficiency, and Mortality in Renal Transplant Recipients Michele F. Eisenga,* Marco van Londen,* David E. Leaf,† Ilja M. Nolte,‡ Gerjan Navis,* Stephan J.L. Bakker,* Martin H. de Borst,* and Carlo A.J.M. Gaillard* *Division of Nephrology, Department of Internal Medicine, and ‡Department of Epidemiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; and †Division of Renal Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts

ABSTRACT Iron deficiency (ID) is independently associated with an increased risk of death in renal transplant recipients (RTRs). ID promotes production and cleavage of intact fibroblast growth factor 23 (iFGF23) into C-terminal fibroblast growth factor 23 (cFGF23), elevated levels of which are also prospectively associated with adverse outcomes. We hypothesized that in RTRs, the relationship between ID and mortality is mediated by FGF23. We measured plasma iFGF23 and cFGF23 levels in 700 stable RTRs at a median of 5.4 years after transplant. RTRs with ID had median (interquartile range) cFGF23 concentrations higher than those of RTRs without ID (223 [131–361] versus 124 [88–180] RU/ml; P,0.001), whereas iFGF23 concentrations were similar between groups. In multivariable-adjusted Cox regression analyses, ID associated with increased mortality (81 events; hazard ratio, 1.95; 95% confidence interval, 1.22 to 3.10; P,0.01). However, this association lost significance after additional adjustment for cFGF23 levels (hazard ratio, 1.45; 95% confidence interval, 0.87 to 2.51; P=0.15). In further mediation analysis, cFGF23 explained 46% of the association between ID and mortality, whereas iFGF23 did not mediate this association. In conclusion, we found that cFGF23 levels are increased in iron-deficient RTRs and that the underlying biologic process driving production and cleavage of iFGF23, or alternatively the increased level of cFGF23 fragments, probably is an important mediator of the association between ID and mortality. Our results underline the strong relationship between iron and FGF23 physiology, and provide a potential mechanism explaining the relationship between ID and adverse outcome in RTRs. J Am Soc Nephrol 28: ccc–ccc, 2017. doi: https://doi.org/10.1681/ASN.2016121350

Iron deficiency (ID) is highly prevalent among renal transplant recipients (RTRs) and an important contributor to post-transplant anemia.1,2 In addition to its role in hemoglobin synthesis, iron also plays a pivotal role in oxygen sensing, synthesis of DNA, electron transport, and cellular proliferation and differentiation.3 Independent of anemia, ID is a known risk factor for mortality in RTRs, although the underlying mechanisms are unclear.4 Recent studies suggest that ID is crucially involved in fibroblast growth factor 23 (FGF23) production and metabolism. FGF23 is an osteocyte-derived hormone, and an essential regulator of phosphate metabolism, among others, by influencing vitamin D homeostasis. J Am Soc Nephrol 28: ccc–ccc, 2017

Higher plasma FGF23 levels are associated with an increased risk of mortality in RTRs,5,6 which may be explained, at least in part, by off-target effects of high

Received December 21, 2016. Accepted June 16, 2017. M.H.d.B. and C.A.J.M.G. are the cosenior authors. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Mr. Michele F. Eisenga, Division of Nephrology, Department of Internal Medicine, University Medical Center Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands. Email: [email protected] Copyright © 2017 by the American Society of Nephrology

ISSN : 1046-6673/2812-ccc

1


www.jasn.org

FGF23 levels on the heart and other organs.7,8 Experimental animal models revealed that ID stimulates FGF23 transcription accompanied by increased intracellular cleavage of FGF23, which results in elevated circulating C-terminal FGF23 (cFGF23) concentrations, but relatively normal intact FGF23 (iFGF23) concentrations.9 Observational studies in humans have also demonstrated an inverse relationship between markers of iron status and cFGF23 levels.10–13 Moreover, administration of intravenous iron in iron-deficient anemic women resulted in markedly decreased circulating cFGF23 concentrations,14 consistent with the notion that ID is an important physiologic regulator of increased cFGF23 levels. To date, it is unknown whether an association exists between FGF23 and ID in RTRs and whether FGF23 modulates the increased mortality risk observed in RTRs with ID. Therefore, the aim of this study was to elucidate whether ID influences FGF23 levels, and whether the association of ID with all-cause mortality is mediated by FGF23.

RESULTS Baseline Characteristics

We included 700 RTRs at a median of 5.4 (interquartile range [IQR], 1.9–12.0) years after transplantation. Mean age was 53613 years; 57% of participants were men; mean body mass index (BMI) was 26.764.8 kg/m2. Additional baseline characteristics are shown in Table 1. Median plasma iFGF23 and cFGF23 concentrations were 62 (IQR, 43–99) pg/ml and 140 (95–233) RU/ml, respectively. Mean hemoglobin concentration was 13.261.7 g/dl; median ferritin concentration was 118 (54–222) mg/L; and mean TSAT was 25.4%610.8%. ID, defined as transferrin saturation (TSAT) ,20% and ferritin,300 mg/L, was present in 208 (30%) patients. Significant differences in baseline characteristics between RTRs with versus without ID were noted with respect to sex, BMI, smoking status, time since transplantation, diabetes mellitus, hemoglobin concentration, mean corpuscular volume, ferritin, TSAT, proteinuria, high-sensitivity Creactive protein (hs-CRP), and use of angiotensin converting enzyme (ACE) inhibitors and diuretics (Table 1). Increased cFGF23 levels were noted in the iron-deficient compared with non–iron-deficient RTRs (223 [131–361] versus 124 [88– 180] RU/ml; P,0.001), whereas iFGF23 levels were similar between groups (Table 1). ID, FGF23, and All-Cause Mortality

During a median follow-up of 3.1 (2.7–3.9) years, 81 (12%) RTRs died, of whom 38 (47%) died from cardiovascular causes. Other causes of death were infection (24%), malignancy (16%), and miscellaneous causes (14%). In unadjusted Cox regression analysis, ID (hazard ratio [HR], 2.04 for present versus absent; 95% confidence interval [95% CI], 1.31 to 3.16; P,0.001), cFGF23 (HR, 1.61 per SD; 95% CI, 1.35 to 1.91; P,0.001), and iFGF23 (HR, 1.33 per SD; 2

Journal of the American Society of Nephrology

95% CI, 1.11 to 1.60; P=0.002) were associated with all-cause mortality. The simulation study showed that this difference in HRs could not be explained by the differences in intra-assay coefficients of variation (CVs) (HR cFGF23, 1.60 per SD; 95% CI, 1.35 to 1.90). We observed no effect modification of the association between ID and mortality by age, sex, eGFR, proteinuria, history of cardiovascular disease, time since transplantation, smoking status, BMI, presence of diabetes, hs-CRP, serum calcium, phosphate, parathyroid hormone (PTH), use of ACE inhibitors, use of diuretics, cFGF23, and iFGF23 concentrations (Pinteraction.0.10 for all). In multivariable Cox regression analysis, the association between ID and mortality remained significant after adjustment for age, sex, eGFR, proteinuria, time since transplantation, primary renal disease, history of cardiovascular disease, and smoking status (HR, 1.95; 95% CI, 1.22 to 3.10; P,0.01). Further adjustment for iFGF23 did not affect the association between ID and mortality (HR, 1.94; 95% CI, 1.22 to 3.10; P,0.01). In contrast, adjustment for cFGF23 abolished the association between ID and mortality such that it was no longer significant (HR, 1.45; 95% CI, 0.87 to 2.51; P=0.15) (Table 2). The correlation coefficient of ID with cFGF23 levels was r=0.35, P,0.001, and the variance inflation factor (VIF) was 1.1, indicating absence of relevant colinearity. Mediation Analysis

In mediation analyses, iFGF23 was not found to be a significant mediator (P value for indirect effect .0.05) of the effect between ID and mortality. In contrast, cFGF23 was a significant mediator (P value for indirect effect ,0.05; 46% of the association between ID and mortality was explained by cFGF23) (Table 3). Differences in intra-assay CVs could not explain this difference in mediation effects either (proportion of mediation from simulated data, 45.7%). Sensitivity Analyses

In sensitivity analyses, we first assessed the prospective association between ID and mortality using an alternative definition of ID: TSAT,20% and ferritin,200 mg/L. The association between ID and mortality remained significant independent of adjustment for age, sex, eGFR, proteinuria, time since transplantation, primary renal disease, history of cardiovascular disease, and smoking status (HR, 1.87; 95% CI, 1.16 to 3.01; P=0.01). Similar to the primarily used definition of ID, the association persisted after adjustment for iFGF23 (HR, 1.87; 95% CI, 1.16 to 3.02; P=0.01), whereas adjustment for cFGF23 abolished the association (HR, 1.40; 95% CI, 0.81 to 2.40; P=0.23). Second, we assessed whether the association between ID and mortality remained independent of adjustment for markers related to mineral metabolism other than FGF23, i.e., serum calcium and serum PTH. In these analyses, the association between ID and mortality remained materially unchanged independent of further adjustment for serum calcium in addition to adjustment for age, sex, eGFR, proteinuria, time since transplantation, primary renal disease, and smoking status (HR, 1.90; 95% CI 1.19 to 3.03; P,0.01). The same was true J Am Soc Nephrol 28: ccc–ccc, 2017

www.jasn.org


Table 1. Baseline characteristics of RTRs according to ID Variables

Total Population (n=700)

No ID (n=492)

ID (n=208)

P Value

Age, yr Men, n (%) BMI, kg/m2 Body surface area, m2 Alcohol use, n (%) Smoking status, n (%) Never smoker Former smoker Current smoker Primary renal disease, n (%) Primary glomerular disease Glomerulonephritis Tubulo-interstitial disease Polycystic renal disease Dysplasia and hypoplasia Renovascular disease Diabetic nephropathy Other or unknown cause History of cardiovascular disease, n (%) Time since transplantation, yr Acute rejection, n (%) Diabetes mellitus, n (%) Systolic BP, mmHg Diastolic BP, mmHg Laboratory measurements iFGF23, pg/ml cFGF23, RU/ml Hemoglobin, g/dl MCV, fL Ferritin, mg/L TSAT, % Total cholesterol, mmol/L Phosphate, mmol/L Calcium, mmol/L PTH, pmol/L eGFR, ml/min per 1.73 m2 Creatinine, mmol/L Proteinuria (.0.5 g), n (%) hs-CRP, mg/L Treatment, n (%) ACE inhibitors b-blocker Calcium channel blockers Diuretic use Oral iron supplements

53613 398 (57) 26.764.8 1.9 (1.8–2.1) 569 (82)

53613 299 (61) 26.364.6 1.9 (1.8–2.1) 393 (80)

54612 99 (48) 27.565.0 2.0 (1.8–2.1) 176 (85)

0.37 0.001 ,0.001 0.15 0.16 0.02

317 (45) 299 (43) 84 (12)

232 (47) 194 (39) 66 (13)

85 (41) 105 (51) 18 (9)

197 (28) 53 (8) 83 (12) 145 (21) 29 (4) 40 (6) 35 (5) 118 (17) 96 (14) 5.4 (1.9–12.0) 186 (27) 170 (24) 136617 83611

132 (27) 40 (8) 63 (13) 98 (20) 23 (5) 27 (6) 20 (4) 89 (18) 54 (11) 6.2 (2.6–13.0) 130 (26) 97 (20) 135617 82611

65 (31) 13 (6) 20 (10) 47 (23) 6 (3) 13 (6) 15 (7) 29 (14) 42 (20) 4.3 (1.1–10.0) 56 (27) 73 (35) 137617 83611

,0.01 ,0.001 0.89 ,0.001 0.23 0.20

62 (43–99) 140 (95–233) 13.261.7 9064 118 (54–222) 25.4610.8 5.161.1 1.060.2 2.4060.15 8.9 (5.9–14.7) 52620 138659 158 (23) 1.6 (0.7–4.6)

62 (42–101) 124 (88–180) 13.561.7 9266 158 (88–267) 30.268.9 5.161.1 1.060.2 2.4160.15 8.7 (6.1–14.4) 53.2620.4 138659 100 (23) 1.4 (0.6–3.5)

62 (49–98) 223 (131–361) 12.761.7 8866 46 (27–93) 13.964.2 5.261.2 1.060.2 2.3960.16 9.7 (5.5–15.6) 50.4619.6 138660 58 (28) 2.5 (1.0–6.3)

0.29 ,0.001 ,0.001 ,0.001 ,0.001 ,0.001 0.57 0.50 0.33 0.49 0.10 0.90 0.03 ,0.001

227 (32) 444 (63) 171 (24) 284 (41) 41 (6)

173 (35) 307 (62) 118 (24) 183 (37) 30 (6)

54 (26) 137 (66) 53 (26) 101 (49) 11 (5)

0.02 0.38 0.67 ,0.01 0.68

0.24

Values are means6SD, medians (IQR), or numbers (%). MCV, mean corpuscular volume.

with further adjustment for serum PTH (HR, 1.96; 95% CI, 1.23 to 3.13; P,0.01 [see Table 2, model 2 for comparison]). Furthermore, we assessed whether the association between ID and mortality remained independent of adjustment for ACE inhibitors and angiotensin II (AII) antagonists, hemoglobin levels, acute rejection, presence of diabetes, use of iron supplements, or hs-CRP. In these analyses, the association between ID and mortality remained materially unchanged independent J Am Soc Nephrol 28: ccc–ccc, 2017

of further adjustment for use of ACE inhibitors and AII antagonists (HR, 1.97; 95% CI, 1.23 to 3.16; P,0.01 [see Table 2, model 2 for comparison]), in addition to adjustment for age, sex, eGFR, proteinuria, time since transplantation, primary renal disease, and smoking status. The same was true with further adjustment for hemoglobin (HR, 1.76; 95% CI, 1.09 to 2.84; P=0.02), acute rejection (HR, 1.93; 95% CI, 1.21 to 3.09; P,0.01), presence of diabetes (HR, 1.83; 95% CI, 1.13 to Iron Deficiency, FGF23, and Renal Transplant

3


www.jasn.org

Table 2. Univariate and multivariate-adjusted associations between ID and all-cause mortality Model

HR (95% CI)

P Value

Univariate Model 1 Model 2 Model 3 Model 4

2.04 (1.31 to 3.16) 1.94 (1.25 to 3.01) 1.95 (1.22 to 3.10) 1.94 (1.22 to 3.10) 1.45 (0.87 to 2.51)

0.001 0.003 ,0.01 ,0.01 0.15

Model 1: adjustment for age and sex; model 2: model 1+adjustment for eGFR, proteinuria, time since transplantation, primary renal disease, history of cardiovascular disease, and smoking status; model 3: model 2+adjustment for iFGF23; model 4: model 2+adjustment for cFGF23; cFGF23, iFGF23, and time since transplantation were ln-transformed before adding to the Cox regression analysis due to skewed distribution.

highest quintile of ferritin was still significantly associated with mortality (HR, 2.78; 95% CI, 1.22 to 6.37; P=0.02; Figure 1B). TSAT was inversely associated with mortality (HR, per 5% increase, 0.86; 95% CI, 0.77 to 0.96; P,0.01; Figure 1C). In multivariable analysis, TSAT remained significantly associated with mortality (HR, 0.87; 95% CI, 0.78 to 0.98; P=0.02). Adjustment for iFGF23 did not alter the association of TSAT with mortality (HR, 0.88; 95% CI, 0.78 to 0.98; P=0.02). In contrast, further adjustment for cFGF23 abolished the association (HR, 0.96; 95% CI, 0.84 to 1.08; P=0.45; Figure 1D).

DISCUSSION

2.94; P=0.01), use of iron supplements (HR, 2.06; 95% CI, 1.29 to 3.29; P=0.003), and hs-CRP (HR, 1.86; 95% CI, 1.15 to 2.99; P=0.01). Finally, we assessed the associations between the individual iron status components and mortality. The relationship between serum ferritin and mortality demonstrated a nonlinear relationship, as shown by cubic restricted splines (Figure 1A). When divided into quintiles and adjusted according to model 2, the lowest and the highest quintiles of serum ferritin were associated with an increased risk of mortality (HR, 2.82; 95% CI, 1.20 to 6.63; P=0.02; and HR, 2.49; 95% CI, 1.10 to 5.65; P=0.03, respectively), compared with the fourth quintile. Adjustment for iFGF23 did not materially alter the association of the lowest quintile (HR, 2.79; 95% CI, 1.19 to 6.59; P=0.02) and the highest quintile (HR, 2.54; 95% CI, 1.12 to 5.77; P=0.03) of ferritin with mortality. In contrast, after adjustment for cFGF23, the association of the lowest quintile with mortality was markedly weakened and no longer significant (HR, 1.67; 95% CI, 0.66 to 4.22; P=0.27), but the

In this study, we show that, in RTRs, ID is accompanied by markedly higher cFGF23 levels than in RTRs without ID, whereas iFGF23 levels were similar in both groups. Importantly, variation in cFGF23 explained a considerable part of the association between ID and mortality. Similar findings were obtained in sensitivity analyses using an alternative definition of ID, and using TSATand ferritin as individual components of ID. Thus, this study confirms earlier findings that iron plays an essential role in FGF23 production and metabolism, extends these findings to a novel patient setting (i.e., RTRs), and supports the notion that the biologic process by which ID simultaneously upregulates FGF23 production and its cleavage, or alternatively the increased level of cFGF23 fragments, is an important mediator of the association between ID and mortality. Post-transplantation anemia is highly prevalent in RTRs, affecting approximately one third of the population.2,4 Furthermore, it has been widely documented that post-transplant

Table 3. Mediation analysis of ID with all-cause mortality through iFGF23, cFGF23, and hs-CRP Outcome

Effect (Path)a

iFGF23

All-cause mortality

cFGF23

All-cause mortality

hs-CRP

All-cause mortality

Indirect effect (ab path) Total effect (ab + c’ path) Unstandardized total effecte Indirect effect (ab path) Total effect (ab + c’ path) Unstandardized total effecte Indirect effect (ab path) Total effect (ab + c’ path) Unstandardized total effecte

Potential Mediator

Multivariable Modelb Coefficient (95% CI, bc)c

Proportion Mediatedd

0.01 (20.01 to 0.01) 0.14 (0.04 to 0.23) 0.80 (0.26 to 1.33) 0.06 (0.02 to 0.11) 0.13 (0.03 to 0.22) 0.48 (20.10 to 1.07) 0.01 (20.01 to 0.04) 0.14 (0.03 to 0.24) 0.77 (0.22 to 1.32)

Not mediated

46%

Not mediated

Bc, bias corrected. a The coefficients of the indirect ab path and the total ab + c’ path are standardized for the SD of the ID, cFGF23, iFGF23, and ratio of cFGF23 to iFGF23 and all-cause mortality. b All coefficients are adjusted for age, sex, eGFR, proteinuria, time since transplantation, primary renal disease, history of cardiovascular disease, and smoking status. c 95% CIs for the indirect and total effects were bias-corrected 95% CIs after running 2000 bootstrap samples. d The size of the significant mediated effect is calculated as the standardized indirect effect divided by the standardized total effect multiplied by 100. e Odds ratios for risk of outcomes can be calculated by taking the exponent of the unstandardized total effect. For example, the unstandardized coefficient of the direct effect of ID on all-cause mortality while adjusting for ratio cFGF23 is 0.48, which can be calculated to an odds ratio by taking the exponent of this regression coefficient, i.e., e0.483=1.62, which comes near the HR of 1.45 (see Table 2). The difference between odds ratio and HR is explained that mediation analysis is on the basis of logistic regression which does not take into account time to event.

4


J Am Soc Nephrol 28: ccc–ccc, 2017

www.jasn.org


ferritin with mortality is supportive of our hypothesis that inflammation is not the driving force behind the association of ID and mortality. In line with this observation, the association of ID with mortality persisted upon adjustment for hs-CRP. Similarly, renal function in itself also does not seem to be the sole underlying factor, because adjustment for eGFR did not materially change the results either in this study or in previous studies addressing the association between cFGF23 and mortality.5,6 As an alternative to an underlying factor or process mediating both FGF23 cleavage and adverse outcome, it may be that C-terminal fragments in themselves drive adverse outcome. Previously, C-terminal fragments have been found to compete with iFGF23 for binding to its receptor complex and function as a competitive inhibitor, which may impair phosphaturia and aggravate soft tissue calcification.21 The present data confirm the concept Figure 1. Associations of low serum ferritin and low TSAT with all-cause mortality are that iron plays an important role in reguabrogated by adjustment for cFGF23. Data were fit by a Cox proportional hazard lating FGF23 production and metabolism. regression model on the basis of restricted cubic splines. For both variables, the FGF23 is regulated by a complex, partly median was utilized as reference (4.8 mg/L for naturally logarithmic ferritin, 24% for unrevealed, interplay between local bone TSAT). The black line represents the HR, the gray area the 95% CI. Panels (A and C) factors that modulate bone turnover and show univariate analyses between serum ferritin and TSAT and all-cause mortality. mineralization, and systemic factors that Panels (B and D) show adjustment for cFGF23. regulate mineral metabolism. 22 PTH, 1,25-dihydroxyvitamin D, klotho, glucocorticoids, calcium, and phosphate are all known to regulate FGF23 production.23,24 It has been established that anemia is associated with poor outcomes.1,15,16 Recently, we showed that ID, independent of anemia, is associated with an systemic ID stabilizes hypoxia-inducible factor 1-a (HIF-1a), increased risk of mortality, shifting the focus from anemia to which increases FGF23 transcription9 and simultaneously upreID.4 The etiology of ID-related risk of mortality is unknown. gulates furin, which cleaves FGF23.25,26 Normal osteocytes couple In this study, we have shown that the association of ID with increased FGF23 production with commensurately increased mortality is largely explained by cFGF23. The extent of this FGF23 cleavage, which ensures normal phosphate homeostasis effect is illustrated by the restricted cubic splines depicting the in the event of ID26 because the intact, biologically active individual components of ID (Figure 1). The association beiFGF23 levels remain relatively unchanged. In keeping with this tween low serum ferritin and low TSAT and mortality is abhypothesis, in our iron-deficient RTRs’ cFGF23 levels were uprerogated by adjustment for cFGF23, whereas the association gulated, whereas the levels of iFGF23 were similar in patients with between high serum ferritin levels and mortality was not altered. ID and without ID. In mediation analyses, cFGF23 was a prominent statistical meTo our knowledge, this is the first study to investigate the diator of the association between ID and mortality. Therefore, it interplay between ID and FGF23 in RTRs. In animal models seems that an ID-induced rise in cFGF23 levels plays an impor- of CKD, it has been shown that ID stimulates FGF23 production tant role in the outcome of RTRs. Indeed, cFGF23 has been but also upregulates cleavage, which leads to increased levels of shown to be an independent risk factor for death in various circulating cFGF23, but normal levels of iFGF23.9,27,28 In hupatient groups, including postoperative AKI, nondialysis CKD, mans, Wolf and colleagues showed that ID anemia is associated ESRD, and RTRs.5,6,17–20 It seems likely that a so far unknown, with normal iFGF23 but markedly elevated cFGF23 levels to an extent that is only seen in advanced CKD or in hereditary disunderlying process driving FGF23 production and cleavage eases of FGF23 overproduction.14 Moreover, rapid correction of plays a role in adverse outcomes of ID, but not iron overload or inflammation. The fact that adjustment for cFGF23 did not ID with different intravenous iron preparations reduced cFGF23 materially alter the upper part of the nonlinear association that levels by approximately 80% within 24 hours,14 consistent with was present in sensitivity analyses on the association of serum ID as an important stimulus for elevated cFGF23 levels. J Am Soc Nephrol 28: ccc–ccc, 2017

Iron Deficiency, FGF23, and Renal Transplant

5


www.jasn.org

Our study has several strengths as well as limitations. The main strength is the large prospectively followed cohort of stable RTRs, in which several markers of ID as well as both iFGF23 and cFGF23 levels were measured. Moreover, end point evaluation was complete in all participants despite a considerable follow-up period. We also acknowledge several limitations of this study. First, because of the observational status of our single center study, we cannot exclude the possibility of residual confounding. Second, a limitation is that no gold standard for the definition of ID exists.29 In this study, the definition of ID was on the basis of a combination of two commonly used and clinically relevant markers, namely ferritin (iron load) and TSAT (iron transport availability). To increase robustness of our findings, we performed sensitivity analyses where we used an alternative definition of ID and also assessed the association of the individual iron status parameters with mortality, and demonstrated similar results. In conclusion, we identified that iron-deficient RTRs have elevated levels of cFGF23, but not iFGF23, compared with non–iron-deficient RTRs. Importantly, ID was independently associated with mortality, and this association was to a large extent explained by variation in cFGF23 levels. Future studies are needed to unravel the complex interplay between ID, FGF23, and adverse outcomes in RTRs and other populations.

CONCISE METHODS Patient Population We approached all RTRs (aged$18 years) who were at least 1 year post-transplantation for participation in this study. RTRs were approached during outpatient clinic visits between 2008 and 2011, as described previously.30 All kidney transplantations took place in the University Medical Center Groningen (Groningen, The Netherlands). Among 817 RTRs who were approached, 707 (87%) chose to participate. We excluded patients with missing data on ID (n=7), resulting in 700 RTRs eligible for analyses. All patients provided written informed consent. All study protocols were approved by the Medical Ethical Committee (METc 2008/186) and adhered to the principles of the Declaration of Helsinki. The primary end point of this study was all-cause mortality. The continuous surveillance system of the outpatient program ensures up-to-date information on patient status. General practitioners or referring nephrologists were contacted in the case that the status of a patient was unknown. End points were recorded until the end of May of 2013 with no loss to follow-up.

Data Collection The measurement of clinical parameters has been described in detail previously.31 In brief, information on medical history and medication use was obtained from patient records. Participants’ height and weight were measured with participants wearing indoor clothing without shoes. BP was measured according to a strict protocol as previously described.31 Information on alcohol consumption and smoking behavior was gathered using a questionnaire. Smoking behavior was classified as never, former, or current smoker. 6


Laboratory Procedures Blood was drawn in the morning after an 8–12-hour overnight fast. Plasma cFGF23 levels were measured with human FGF23 (C-terminal) ELISA (Immutopics, Inc., San Clemente, CA) in stored plasma samples with intra-assay CVs between 2.2% and 4.4%, and interassay CVs between 9% and 16%.32 iFGF23 levels were measured in stored plasma samples by ELISA (Kainos Laboratories, Inc., Tokyo, Japan) with intraassay CVs between 5.3% and 9.7%, and interassay CVs between 5.7% and 14%.32 The cFGF23 immunometric assay uses two antibodies directed against different epitopes within the C-terminal part of FGF23, which therefore detects both the intact hormone and C-terminal cleavage products. In contrast, the iFGF23 assay detects only the intact molecule.26 Transferrin was measured using an immunoturbidimetric assay (Cobas c analyzer, Modular P system; Roche Diagnostics, Mannheim, Germany). Serum ferritin concentrations were determined using the electrochemiluminescence immunoassay (Modular analytics E170; Roche Diagnostics). Serum iron was measured using photometry (Modular P800 system; Roche Diagnostics). Serum creatinine was measured using an enzymatic, isotope dilution mass spectrometry–traceable assay on a Roche P-Modular automated analyzer (Roche Diagnostics). TSAT (%) was calculated as 1003 serum iron (mmol/L)/253 transferrin (g/L).33 ID was defined as TSAT,20% and ferritin,300 mg/L.34 Renal function was determined by estimating GFR by applying the CKD Epidemiology Collaboration equation.35 Proteinuria was defined as urinary protein excretion $0.5 g/24 h.

Statistical Analyses Data were analyzed using IBM SPSS software, version 22.0 (SPSS Inc., Chicago, IL), R version 3.2.3 (Vienna, Austria), and STATA 14.1 (STATA Corp., College Station, TX). Data are expressed as mean6 SD for normally distributed variables and as median (25th–75th IQR) for variables with a skewed distribution. Categoric data are expressed as number (percentage). We evaluated between-group differences comparing RTRs with versus without ID using t test, Mann–Whitney U test, or chi-squared test, as appropriate. To study the association between ID and all-cause mortality, we used Cox proportional hazards regression analysis. We performed analyses in which we first adjusted for age and sex (model 1); and additionally for eGFR, proteinuria, time since transplantation, primary renal disease, history of cardiovascular disease, and smoking status (model 2). In further models, we adjusted for iFGF23 (model 3) and cFGF23 (model 4). Because of skewed distribution, iFGF23, cFGF23, and time since transplantation were natural log–transformed. We tested for colinearity between ID and cFGF23 by calculating a correlation coefficient and a VIF score. A correlation coefficient of ,0.7 and a VIF,5 indicate no evidence for colinearity.36 Potential effect modifications by age, sex, eGFR, proteinuria, history of cardiovascular disease, time since transplantation, smoking status, BMI, presence of diabetes, hs-CRP, serum calcium, phosphate, PTH, use of ACE-inhibitors, use of diuretics, cFGF23, and iFGF23 concentrations were tested by fitting models containing both main effects and their crossproduct terms. In sensitivity analyses, we repeated the Cox regression analysis by using an alternative, frequently used definition of ID: TSAT,20% and ferritin,200 mg/L.37 We also evaluated ID using the iron status components (i.e., TSAT and ferritin) individually. Splines of individual iron status components with J Am Soc Nephrol 28: ccc–ccc, 2017

www.jasn.org

all-cause mortality were fit using a Cox proportional hazards regression model on the basis of restricted cubic splines in univariate analyses and after adjustment for cFGF23. As sensitivity analyses, we performed adjustments of the association of ID with mortality as in Table 2, model 2, for serum calcium, serum PTH, use of ACE inhibitors and AII antagonists, hemoglobin levels, acute rejection events, presence of diabetes, use of iron supplements, and hs-CRP levels, each time in addition to existing adjustment for age, sex, eGFR, proteinuria, time since transplantation, primary renal disease, and smoking status. Finally, we performed mediation analyses with the method described by Preacher and Hayes, which is on the basis of logistic regression. These analyses allow for testing significance and magnitude of mediation.38,39 In all analyses, a two-sided P value ,0.05 was considered significant.

Simulation Study In order to investigate the effect of differences in intra-assay CVs between cFG23 and iFGF23 we simulated values of cFGF23 100 times. In each simulation, normally distributed noise with a mean of 0 and an SD equal to 4.3% of the cFGF23 value was added to the original cFGF23 value in order to simulate an intra-assay CV between 9.5% and 10%, which is similar to that of iFGF23. The Cox regression and the mediation analyses were repeated for each of these simulated cFGF23 variables and the means of the HRs, their 95%CIs, and the proportionof mediationwere calculated.

ACKNOWLEDGMENTS We thank Wendy Dam for measuring cFGF23 levels in the plasma samples of the cohort. Generation of the TransplantLines Food and Nutrition Biobank and Cohort Study (TxL-FN) was funded by Top Institute Food and Nutrition. M.H.d.B. is supported by a Veni grant (The Netherlands Organization for Scientific Research, grant no 016.146.014).

6.

7.

8.

9.

10.

11.

12.

13.

14.

DISCLOSURES

15.

C.A.J.M.G. received speaking fees and research funding from Vifor Pharma. The other authors have declared that no conflict of interest exists. 16.

REFERENCES 1. Imoagene-Oyedeji AE, Rosas SE, Doyle AM, Goral S, Bloom RD: Posttransplantation anemia at 12 months in kidney recipients treated with mycophenolate mofetil: Risk factors and implications for mortality. J Am Soc Nephrol 17: 3240–3247, 2006 2. Lorenz M, Kletzmayr J, Perschl A, Furrer A, Hörl WH, Sunder-Plassmann G: Anemia and iron deficiencies among long-term renal transplant recipients. J Am Soc Nephrol 13: 794–797, 2002 3. Lieu PT, Heiskala M, Peterson PA, Yang Y: The roles of iron in health and disease. Mol Aspects Med 22: 1–87, 2001 4. Eisenga MF, Minovic I, Berger SP, Kootstra-Ros JE, van den Berg E, Riphagen IJ, Navis G, van der Meer P, Bakker SJ, Gaillard CA: Iron deficiency, anemia, and mortality in renal transplant recipients. Transpl Int 29: 1176–1183, 2016 5. Wolf M, Molnar MZ, Amaral AP, Czira ME, Rudas A, Ujszaszi A, Kiss I, Rosivall L, Kosa J, Lakatos P, Kovesdy CP, Mucsi I: Elevated fibroblast


17.

18.

19.

20.


growth factor 23 is a risk factor for kidney transplant loss and mortality. J Am Soc Nephrol 22: 956–966, 2011 Baia LC, Humalda JK, Vervloet MG, Navis G, Bakker SJ, de Borst MH; NIGRAM Consortium: Fibroblast growth factor 23 and cardiovascular mortality after kidney transplantation. Clin J Am Soc Nephrol 8: 1968– 1978, 2013 Faul C, Amaral AP, Oskouei B, Hu MC, Sloan A, Isakova T, Gutiérrez OM, Aguillon-Prada R, Lincoln J, Hare JM, Mundel P, Morales A, Scialla J, Fischer M, Soliman EZ, Chen J, Go AS, Rosas SE, Nessel L, Townsend RR, Feldman HI, St John Sutton M, Ojo A, Gadegbeku C, Di Marco GS, Reuter S, Kentrup D, Tiemann K, Brand M, Hill JA, Moe OW, Kuro-O M, Kusek JW, Keane MG, Wolf M: FGF23 induces left ventricular hypertrophy. J Clin Invest 121: 4393–4408, 2011 Rossaint J, Oehmichen J, Van Aken H, Reuter S, Pavenstädt HJ, Meersch M, Unruh M, Zarbock A: FGF23 signaling impairs neutrophil recruitment and host defense during CKD. J Clin Invest 126: 962–974, 2016 Farrow EG, Yu X, Summers LJ, Davis SI, Fleet JC, Allen MR, Robling AG, Stayrook KR, Jideonwo V, Magers MJ, Garringer HJ, Vidal R, Chan RJ, Goodwin CB, Hui SL, Peacock M, White KE: Iron deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) phenotype in fibroblast growth factor-23 (Fgf23) knock-in mice. Proc Natl Acad Sci U S A 108: E1146–E1155, 2011 Durham BH, Joseph F, Bailey LM, Fraser WD: The association of circulating ferritin with serum concentrations of fibroblast growth factor-23 measured by three commercial assays. Ann Clin Biochem 44: 463–466, 2007 Braithwaite V, Prentice AM, Doherty C, Prentice A: FGF23 is correlated with iron status but not with inflammation and decreases after iron supplementation: A supplementation study. Int J Pediatr Endocrinol 2012: 27, 2012 Imel EA, Peacock M, Gray AK, Padgett LR, Hui SL, Econs MJ: Iron modifies plasma FGF23 differently in autosomal dominant hypophosphatemic rickets and healthy humans. J Clin Endocrinol Metab 96: 3541–3549, 2011 van Breda F, Emans ME, van der Putten K, Braam B, van Ittersum FJ, Kraaijenhagen RJ, de Borst MH, Vervloet M, Gaillard CA: Relation between red cell distribution width and fibroblast growth factor 23 cleaving in patients with chronic kidney disease and heart failure. PLoS One 10: e0128994, 2015 Wolf M, Koch TA, Bregman DB: Effects of iron deficiency anemia and its treatment on fibroblast growth factor 23 and phosphate homeostasis in women. J Bone Miner Res 28: 1793–1803, 2013 Chhabra D, Grafals M, Skaro AI, Parker M, Gallon L: Impact of anemia after renal transplantation on patient and graft survival and on rate of acute rejection. Clin J Am Soc Nephrol 3: 1168–1174, 2008 Stoumpos S, Jardine AG, Mark PB: Cardiovascular morbidity and mortality after kidney transplantation. Transpl Int 28: 10–21, 2015 Gutiérrez OM, Mannstadt M, Isakova T, Rauh-Hain JA, Tamez H, Shah A, Smith K, Lee H, Thadhani R, Jüppner H, Wolf M: Fibroblast growth factor 23 and mortality among patients undergoing hemodialysis. N Engl J Med 359: 584–592, 2008 Isakova T, Xie H, Yang W, Xie D, Anderson AH, Scialla J, Wahl P, Gutiérrez OM, Steigerwalt S, He J, Schwartz S, Lo J, Ojo A, Sondheimer J, Hsu CY, Lash J, Leonard M, Kusek JW, Feldman HI, Wolf M; Chronic Renal Insufficiency Cohort (CRIC) Study Group: Fibroblast growth factor 23 and risks of mortality and end-stage renal disease in patients with chronic kidney disease. JAMA 305: 2432–2439, 2011 Kendrick J, Cheung AK, Kaufman JS, Greene T, Roberts WL, Smits G, Chonchol M; HOST Investigators: FGF-23 associates with death, cardiovascular events, and initiation of chronic dialysis. J Am Soc Nephrol 22: 1913–1922, 2011 Leaf DE, Christov M, Jüppner H, Siew E, Ikizler TA, Bian A, Chen G, Sabbisetti VS, Bonventre JV, Cai X, Wolf M, Waikar SS: Fibroblast growth factor 23 levels are elevated and associated with severe acute kidney injury and death following cardiac surgery. Kidney Int 89: 939–948, 2016

Iron Deficiency, FGF23, and Renal Transplant

7


www.jasn.org

21. Goetz R, Nakada Y, Hu MC, Kurosu H, Wang L, Nakatani T, Shi M, Eliseenkova AV, Razzaque MS, Moe OW, Kuro-o M, Mohammadi M: Isolated C-terminal tail of FGF23 alleviates hypophosphatemia by inhibiting FGF23-FGFR-Klotho complex formation. Proc Natl Acad Sci U S A 107: 407–412, 2010 22. Liu S, Tang W, Zhou J, Stubbs JR, Luo Q, Pi M, Quarles LD: Fibroblast growth factor 23 is a counter-regulatory phosphaturic hormone for vitamin D. J Am Soc Nephrol 17: 1305–1315, 2006 23. Kovesdy CP, Quarles LD: Fibroblast growth factor-23: What we know, what we don’t know, and what we need to know. Nephrol Dial Transplant 28: 2228–2236, 2013 24. Nguyen-Yamamoto L, Karaplis AC, St-Arnaud R, Goltzman D: Fibroblast growth factor 23 regulation by systemic and local osteoblastsynthesized 1,25-dihydroxyvitamin D. J Am Soc Nephrol 28: 586–597, 2017 25. McMahon S, Grondin F, McDonald PP, Richard DE, Dubois CM: Hypoxiaenhanced expression of the proprotein convertase furin is mediated by hypoxia-inducible factor-1: Impact on the bioactivation of proproteins. J Biol Chem 280: 6561–6569, 2005 26. Wolf M, White KE: Coupling fibroblast growth factor 23 production and cleavage: iron deficiency, rickets, and kidney disease. Curr Opin Nephrol Hypertens 23: 411–419, 2014 27. Hanudel MR, Chua K, Rappaport M, Gabayan V, Valore E, Goltzman D, Ganz T, Nemeth E, Salusky IB: Effects of dietary iron intake and chronic kidney disease on fibroblast growth factor 23 metabolism in wild type and hepcidin knockout mice. Am J Physiol Renal Physiol 311: F1369– F1377, 2016 28. David V, Martin A, Isakova T, Spaulding C, Qi L, Ramirez V, ZumbrennenBullough KB, Sun CC, Lin HY, Babitt JL, Wolf M: Inflammation and functional iron deficiency regulate fibroblast growth factor 23 production. Kidney Int 89: 135–146, 2016 29. Eisenga MF, Bakker SJ, Gaillard CA: Definition of functional iron deficiency and intravenous iron supplementation. Lancet Haematol 3: e504, 2016 30. van den Berg E, Engberink MF, Brink EJ, van Baak MA, Joosten MM, Gans RO, Navis G, Bakker SJ: Dietary acid load and metabolic acidosis

8


31.

32.

33.

34.

35.

36.

37.

38.

39.

in renal transplant recipients. Clin J Am Soc Nephrol 7: 1811–1818, 2012 van den Berg E, Pasch A, Westendorp WH, Navis G, Brink EJ, Gans RO, van Goor H, Bakker SJ: Urinary sulfur metabolites associate with a favorable cardiovascular risk profile and survival benefit in renal transplant recipients. J Am Soc Nephrol 25: 1303–1312, 2014 Heijboer AC, Levitus M, Vervloet MG, Lips P, ter Wee PM, Dijstelbloem HM, Blankenstein MA: Determination of fibroblast growth factor 23. Ann Clin Biochem 46: 338–340, 2009 Mercadal L, Metzger M, Haymann JP, Thervet E, Boffa JJ, Flamant M, Vrtovsnik F, Gauci C, Froissart M, Stengel B; NephroTest Study Group: A 3-marker index improves the identification of iron disorders in CKD anaemia. PLoS One 9: e84144, 2014 Charytan C, Levin N, Al-Saloum M, Hafeez T, Gagnon S, Van Wyck DB: Efficacy and safety of iron sucrose for iron deficiency in patients with dialysis-associated anemia: North American clinical trial. Am J Kidney Dis 37: 300–307, 2001 Levey AS, Stevens LA, Schmid CH, Zhang YL, Castro AF 3rd, Feldman HI, Kusek JW, Eggers P, Van Lente F, Greene T, Coresh J; CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration): A new equation to estimate glomerular filtration rate. Ann Intern Med 150: 604–612, 2009 Dormann CF, Elith J, Bacher S, Buchmann C, Carl G, Carré G, Marquéz JRG, Gruber B, Lafourcade B, Leitão PJ, Münkemüller T, McClean C, Osborne PE, Reineking B, Schröder B, Skidmore AK, Zurell D, Lautenbach S: Collinearity: A review of methods to deal with it and a simulation study evaluating their performance. Ecography 36: 27–46, 2013 Macdougall IC, Bock A, Carrera F, Eckardt KU, Gaillard C, Van Wyck D, Roubert B, Cushway T, Roger SD; FIND-CKD Study Investigators: The FIND-CKD study–a randomized controlled trial of intravenous iron versus oral iron in non-dialysis chronic kidney disease patients: background and rationale. Nephrol Dial Transplant 29: 843–850, 2014 Preacher K, Hayes A: SPSS and SAS procedures for estimating indirect effects in simple mediation models. Behav Res Methods Instrum Comput. 2004;36(4):717–731 Hayes A: Beyond baron and kenny: Statistical mediation analysis in the new millennium. Commun Monogr. 2009;76(4): 408-420
