Fibroblast Growth Factor 23 and Klotho in AKI

Fibroblast Growth Factor 23 and Klotho in AKI D1X XMarta Christov, MD, PhD, D2X X* D3X XJavier A. Neyra, MD, MSCS, D4X†,‡ X D5X XSanjeev Gupta, MD, D6X X* and D7X XDavid E. Leaf, MD, MMScD§8X X Summary: Acute kidney injury (AKI) is associated with many of the same mineral metabolite abnormalities that are observed in chronic kidney disease. These include increased circulating levels of the osteocyte-derived, vitamin D −regulating hormone, fibroblast growth factor 23 (FGF23), and decreased renal expression of klotho, the co-receptor for FGF23. Recent data have indicated that increased FGF23 and decreased klotho levels in the blood and urine could serve as novel predictive biomarkers of incident AKI, or as novel prognostic biomarkers of adverse outcomes in patients with established AKI. In addition, because FGF23 and klotho exert numerous classic as well as off-target effects on a variety of organ systems, targeting their dysregulation in AKI may represent a unique opportunity for therapeutic intervention. We review the pathophysiology, kinetics, and regulation of FGF23 and klotho in animal and human studies of AKI, and we discuss the challenges and opportunities involved in targeting FGF23 and klotho therapeutically. Semin Nephrol 39:57−75 Ó 2018 Published by Elsevier Inc. Keywords: FGF23, klotho, AKI, mineral metabolism

T

he osteocyte-derived hormone, fibroblast growth factor 23 (FGF23), initially was identified as the causative agent of two rare disorders of urinary phosphate wasting: autosomal-dominant hypophosphatemic rickets and tumor-induced osteomalacia.1,2

OVERVIEW OF MINERAL METABOLISM IN HEALTH AND CHRONIC KIDNEY DISEASE: FOCUS ON FGF23 AND KLOTHO FGF23: Function and Regulators FGF23 Function

FGF23 increases urinary phosphate excretion by reducing expression of the sodium-phosphate co-transporters, NaPi2a and NaPi2c, expressed on the apical surface of the *Department of Medicine, New York Medical College, Valhalla, NY yDivision of Nephrology, Bone and Mineral Metabolism, Department of Internal Medicine, University of Kentucky, Lexington, KY zDivision of Nephrology, Department of Internal Medicine, University of Texas Southwestern, Dallas, TX xDivision of Renal Medicine, Brigham and Women’s Hospital, Boston, MA M.C. and J.A.N. contributed equally to this manuscript Financial support: Supported by grants K23DK106448 (D.E.L.) and K08DK093608 (M.C.) from the National Institute of Diabetes and Digestive Kidney Diseases; by Early Career pilot grant UL1TR001998 (J.A.N.) from the National Center for Advancing Translational Sciences, National Institutes of Health; by the New York Community Trust (M.C.); and by an American Society of Nephrology Foundation for Kidney Research Carl W. Gottschalk Research Scholar Grant (D.E.L.). Conflict of interest statement: none. Address reprint requests to Marta Christov, MD, PhD, Division of Nephrology, Department of Medicine, New York Medical College, Valhalla, NY 10595. E-mail: [email protected] 0270-9295/ - see front matter © 2018 Published by Elsevier Inc. https://doi.org/10.1016/j.semnephrol.2018.10.005

Seminars in Nephrology, Vol 39, No 1, January 2019, pp 57−75

renal proximal tubular cells, and it decreases circulating 1,25-dihydroxyvitamin D (1,25D) levels by reducing expression of the 1a-hydroxylase cytochrome P450 (CYP) enzyme, CYP27B1, which is also expressed in renal proximal tubular cells.3 FGF23 may also decrease circulating 1,25D levels by increasing the renal expression of the catabolic 24-hydroxylase enzyme, CYP24A1.3 In addition, FGF23 can increase distal sodium reabsorption by increasing the expression of the sodium chloride cotransporter, NCC, and it increases calcium absorption by increasing the expression of transient receptor potential cation channel subfamily V member 5 channels in the distal tubule.4,5 In states of excess circulating FGF23 levels and normal kidney function, humans and animals develop hyperphosphaturia, hypophosphatemia, and rickets,2,6 whereas in states of diminished FGF23 levels or function, humans and animals develop increased circulating levels of phosphate and 1,25D, and ectopic soft-tissue and vascular calcification.7-9 FGF23 signaling

FGF23 binds to single-pass transmembrane FGF receptors (FGFRs), which are expressed in a variety of tissues.10 There are four different FGFR isoforms (FGFR1-4). In addition, alternative splicing of FGFR1-3 produces two subtypes: designated as b and c splice variants. The mineral metabolism actions of FGF23 are mediated through FGFR1, which is expressed ubiquitously.11,12 The binding affinity of FGF23 to FGFR1 is enhanced by its co-receptor, klotho (discussed further in the “Klotho: Function and Regulators” section).13 It has long been thought that the differential expression of klotho, a single transmembrane domain protein expressed in the kidneys and parathyroid glands, provides the site specificity of FGF23 action. More recent work has suggested that soluble or secreted klotho can function to provide the scaffolding for FGF23 and FGFR1 binding, thus 57

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potentially expanding the number of tissues that can be targets of FGF23 action.14 In addition, FGF23 has been shown to signal through another FGF receptor, FGFR4, in the heart and liver in a klotho-independent manner.15,16 When FGF23 signaling occurs through FGFR1, downstream targets are affected through the MAP-kinase pathway, whereas FGF23 signaling through FGFR4 occurs via the phospholipase C Ɣ pathway. FGF23 appears to be involved in several pathways independent of mineral metabolism regulation, and its list of off-target effects is still growing. Regulators of FGF23 production

FGF23 consists of 251 amino acids, including a 24 amino acid signal peptide. FGF23 production is increased by high blood phosphate levels or dietary phosphate loading, 1,25D, and numerous other stimuli (Fig. 1). Most studies found that parathyroid hormone (PTH) increased FGF23 production (reviewed by Bergwitz and J€ uppner17), although some studies found that acute PTH treatment initially may decrease circulating FGF23 levels in both humans and mice.18,19 Recent reports have implicated iron deficiency and hypoxia as additional factors that up-regulate FGF23 production; however, the underlying mechanisms remain incompletely understood.20,21 Chronic inflammation, possibly through an iron-deficiency/hypoxia axis, also increases

1,25D PTH Phosphate Iron deficiency Hypoxia Inflammation Erythropoietin Metabolic acidosis Leptin Estrogen

1,25D Statins RAAS blockers PPAR-γ agonists

FGF23 production.22 Both oral and intravenous iron supplementation have been shown to reduce plasma FGF23 levels in patients with iron-deficiency anemia in the setting of both normal and reduced renal function.23,24 Sites of FGF23 production

Early studies have shown Fgf23 messenger RNA (mRNA) expression in the heart, liver, thyroid/parathyroid, small intestine, skeletal muscle, and fetal chondrocytes, as well as lymph nodes.1,2 Subsequently, Fgf23 expression was documented in osteocytes, especially in the case of animals or humans with chronic kidney disease (CKD), where osteocytes are believed to be the major source of increased circulating FGF23 levels.25 Recent work also has shown Fgf23 mRNA expression in liver, kidneys, heart, spleen, and bone marrow.26-30 FGF23 metabolism

An important step in the regulation of FGF23 metabolism is post-translational modification of the cleavage of bioactive intact FGF23 into C-terminal and N-terminal fragments (Fig. 2). FGF23 undergoes cleavage between arginine 179 and serine 180, located in a conserved cleavage site R176XXR179. Cleavage of FGF23 at this site is down-regulated by O-glycosylation of tyrosine

FGF23

Iron PHEX DMP1 Adiponectin

Klotho

Aging Inflammation Oxidative stress Phosphate Uremic toxins RAAS Low 1,25D

Figure 1. Overview of FGF23 and klotho regulation. FGF23 production is stimulated by a number of factors, including PTH, 1,25D, phosphate, iron deficiency, hypoxia, inflammation, erythropoietin, metabolic acidosis, leptin, and estrogen. Conversely, FGF23 production is inhibited by iron supplementation, and in states of phosphate depletion or depravation. Other local regulators of FGF23 production include adiponectin, PHEX, and DMP1. Klotho expression decreases with aging and in states of inflammation, oxidative stress, hyperphosphatemia, uremia, low 1,25D, or activation of the RAAS. Conversely, klotho expression is increased by 1,25D, RAAS blockers, statins, and PPAR-g agonists. FGF23 suppresses klotho expression, and klotho up-regulates FGF23 production in bone, completing a classic negative endocrine feedback loop. The regulators of FGF23 and klotho depicted in this diagram are mostly based on preclinical data. Abbreviations: DMP1, dentrin matrix acidic phosphoprotein 1; PHEX, phosphate-regulating gene with homologies to endopeptidases on the X chromosome; PPAR-g, peroxisome proliferatoractivated receptor g; RAAS, renin-angiotensin-aldosterone system.

59

FGF23 and klotho

residue 178.31 Cleavage at the RXXR site generates a Cterminal 12-kDa and an N-terminal 18-kDa fragment from the 30- to 32-kDa intact, glycosylated FGF23 protein. Additional post-translational modification of FGF23 occurs by phosphorylation of serine 180, adjacent to the RXXR cleavage site, through the kinase FAM20C. Phosphorylation of this residue inhibits glycosylation, thus enhancing FGF23 cleavage (Fig. 2).32 Circulating FGF23 levels comprise the bioactive intact hormone (iFGF23) and N-terminal and C-terminal fragments, although at physiological concentrations the hormonal fragments likely do not contribute to mineral ion regulation. At supraphysiological concentrations the C-terminal fragment can interfere with FGF23 action, both in the kidney and in the bone marrow, by competing with iFGF23 for the FGF23 receptor.33,34 However, in a fibroblast cell line, C-terminal FGF23 did not show any biological activity.35 In states of iron deficiency and acute inflammation, there was increased production of FGF23, which was matched by a commensurate (or near-commensurate) increase in FGF23 cleavage, such that there are high levels of FGF23 fragments in the circulation but normal (or only mildly increased) levels of iFGF23. In contrast, in CKD and end-stage renal disease (ESRD) there is a presumed down-regulation of the cleavage mechanism, and iFGF23 accounts for a larger proportion of the total circulating levels of FGF23.36,37 Two main assays are used to measure circulating FGF23 levels. The immunometric assay for C-terminal FGF23 (cFGF23) detects both C-terminal cleavage fragments as well as the fulllength intact peptide, whereas the assay for iFGF23 measures intact FGF23 only. The primary mode of clearance of FGF23 and its fragments from the circulation is not well understood, although several groups have reported that FGF23 is detectable in the urine.38-40 Mace et al41 measured differences in intact FGF23 between the renal artery and vein of rats, and reported that the extraction ratio was 0.3, indicating that iFGF23 levels in the vein were 30% lower compared with levels in the artery. These findings are consistent with a report in patients with CKD, in which the extraction of FGF23 was approximately 17%, although varied by renal function, with higher extraction in patients with higher glomerular filtration rates.42 These findings suggest that the kidneys participate in clearance of FGF23, either through filtration and/or catabolism. Accordingly, this mode of clearance may be impaired in patients with acutely or chronically reduced kidney function. In addition, FGF23 cleavage by plasminogen activators recently was reported in vitro using purified proteins, although it is not clear if tissue (or urine) plasminogen activators participate in cleavage of FGF23 in the circulation (or urine) under physiological conditions, and how this may be altered in states of kidney injury.43

FGF23 in CKD Although initially identified in rare genetic and acquired hypophosphatemic disorders, FGF23 has since emerged as a strong and independent predictor of morbidity and mortality in diverse populations, particularly in patients with CKD and ESRD.44,45 In CKD and ESRD, plasma FGF23 levels are increased, at times 1,000-fold higher than in normal healthy patients. The increased morbidity associated with FGF23 may be due to its off-target effects, including left ventricular hypertrophy, inflammation, immune dysfunction, bone loss, and inhibition of erythropoiesis leading to anemia.16,46-48 Efforts to decrease FGF23 in CKD patients by controlling phosphate or PTH, however, have met with mixed success, suggesting that additional mechanisms regulate its production.49-52

FGF23 in Other States of Chronic Injury Recent reports have shown that in states of injury or inflammation other tissues aside from bone can produce FGF23. For example, the heart was shown to express Fgf23 mRNA in a model of acute myocardial infarction, the liver was shown to express Fgf23 mRNA in a liver fibrosis model, and the kidneys were shown to produce both Fgf23 mRNA and protein in a number of chronic injury animal models.26,28,35,53 Leaf et al54 also showed FGF23 protein expression by immunohistochemistry performed on colonic adenocarcinoma tissue obtained from a patient with stage IV colon cancer who presented with oncogenic osteomalacia. In each of these models and settings, the pleiotropic production of FGF23 was linked to increased circulating FGF23 levels, although the significance of this production in mediating the adverse effects of increased FGF23 is unknown.

Klotho: Function and Regulators Klotho function

aKlotho (referred to hereafter as klotho) was discovered in 1997 as a gene linked to aging.55 Klotho plays an important role in regulating mineral metabolism homeostasis. Specifically, klotho decreases renal phosphate reabsorption by acting as a co-receptor for FGF23 binding to FGFR1.13 Klotho also directly can promote the internalization and degradation of the NaPi2a cotransporter in the renal proximal tubules.56 Klotho also may be a suppressor of vitamin D signaling,57,58 because klotho knockout mice can be rescued from a phenotype of soft-tissue calcification by deletion of the CYP27B1 gene.59 Finally, klotho has myriad pleotropic actions as an inhibitor of apoptosis, fibrosis, and cell senescence, and as an up-regulator of autophagy.60-63

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Pro-FGF23

N

C 1

25

251 Glycosylation Phosphorylation

Intact FGF23

C

N 25

251 Cleavage

FGF23 fragments

N-fragment

25

179 C-fragment

180

251

Figure 2. Regulation of cleavage of FGF23. FGF23 is produced as a 251 amino acid pro-peptide. A 24 amino acid N-terminal signaling peptide (yellow bar) is cleaved to form the intact hormone. Intact FGF23 underdoes post-translational modification via phosphorylation or glycosylation at a conserved site, which promotes or inhibits cleavage, respectively, into N-terminal (blue bar) and C-terminal (red bar) fragments.

Regulators of klotho production

Klotho expression is down-regulated by a variety of stimuli, including inflammatory cytokines, uremic toxins (eg, indoxyl sulfate and p-cresyl sulfate), hyperphosphatemia, low 1,25D, oxidative stress, and by activation of the renin-angiotensin-aldosterone system (Fig. 1).64 Klotho expression is up-regulated by peroxisome proliferator-activated receptor-g agonists,65-67 renin-angiotensinaldosterone system blockers,68,69 and statins.70 The mechanisms by which these various molecules affect klotho expression include promoter hypermethylation and hyperacetylation.71-73

FGF23 expression in the bone86 or act as a nonenzymatic scaffold protein that enhances FGF23 signaling.14 Soluble klotho is not filtered by the glomerulus but appears to

Klotho expression and metabolism

Klotho is expressed predominantly in the kidney, specifically in the distal tubules, and, to a lesser degree, in the proximal tubules.74 Klotho is also expressed in other organs such as the brain, pancreas, and parathyroid glands.55 The klotho gene encodes a 130-kDa singlepass transmembrane protein that consists of two extracellular domains (KL1 and KL2), a transmembrane domain, and a short cytoplastic tail. Part of the extracellular domain of transmembrane klotho is cleaved at different sites by proteases.75-77 A secreted form of klotho can also be generated from an alternatively spliced transcript.78,79 Cleaved and secreted klotho are released into the circulation and are referred to as soluble klotho. Soluble klotho levels measured in the blood and urine are thought to be derived primarily from cleaved, rather than secreted, klotho.74,80 Soluble klotho acts as an endocrine or paracrine factor that potentially could affect multiple distant organs such as the bone, brain, heart, lungs, and endothelium.81-87 Soluble klotho may also increase

Figure 3. Relative levels and expression of FGF23 and klotho. Patients with normal kidney function have low (physiologic) circulating levels of cFGF23 and iFGF23 and high kidney expression of klotho. As CKD advances to ESRD, circulating levels of cFGF23 and iFGF23 progressively increase, and renal klotho expression (and possibly soluble levels in the circulation and urine) decreases. Patients with AKI have very high circulating levels of cFGF23, but only moderately increased iFGF23, consistent with increases in both FGF23 production and cleavage. Patients with AKI also have very low renal expression of klotho (and low soluble levels the serum and urine in some reports), similar to patients with ESRD. Abbreviations: ESRD, end-stage renal disease.

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FGF23 and klotho

traffic across the proximal tubules, from the basolateral to the luminal side, and then is excreted in the urine.74 Klotho in CKD Renal klotho expression and soluble levels of klotho in the blood and urine are decreased in animals and humans with CKD resulting from a variety of etiologies, including glomerular and tubulointerstitial diseases, diabetic nephropathy, subtotal nephrectomy, and others (reviewed by Neyra and Hu58). Because the kidneys are the primary source of soluble klotho, it is perhaps unsurprising that circulating levels of soluble klotho decrease as CKD progresses. However, the mechanisms of decreased renal klotho expression and decreased levels of soluble klotho that occur in CKD are unknown. Cardiovascular disease (CVD) is a well-recognized and common complication of advanced CKD.88,89 Putative nontraditional risk factors for CVD in CKD are the increase in FGF23 and/or the decrease in soluble klotho levels.16,90 In rodents, hyperphosphatemia and lower soluble klotho levels have been associated with more severe cardiac hypertrophy and fibrosis.91 Interestingly, increased levels of FGF23 were associated with pathologic cardiac remodeling only if klotho deficiency was also present. However, in a different experiment FGF23, independent of klotho, induced cardiac hypertrophy through FGFR4-mediated phospholipase C-g signaling activation in cardiac myocytes.90 Similarly, it has been postulated that klotho, independent of FGF23, may protect the heart against cardiac hypertrophy through inhibition (via blockade of exocytosis) of transient receptor potential canonical 6 currents in cardiomyocytes.92 Finally, klotho supplementation may protect against indoxyl sulfate-mediated cardiac hypertrophy, as shown in mice.93

INCREASED FGF23 IN AKI Overview Similar to the changes that occur in FGF23 and klotho in CKD, expression of these hormones is abnormal in acute kidney injury (AKI) as well, including increased circulating levels of FGF23 and decreased expression of

Table 1. Summary of Changes in FGF23 and Klotho in AKI FGF23 levels increase in the circulation and urine FGF23 mRNA and protein increase in bone FGF23 mRNA increases in some extraskeletal tissues, including the bone marrow and kidney Klotho mRNA expression and protein levels decrease in the kidney Klotho levels likely decrease in the circulation and urine

klotho in the kidney (and possibly decreased soluble klotho levels in blood and urine). These changes are summarized in Table 1 and Figure 3. Abnormalities in other mineral metabolites in AKI, including calcium, phosphate, PTH, and vitamin D, are discussed in detail in Leaf and Christov94 in this issue. FGF23 is Increased in Human AKI The first evidence that circulating FGF23 levels are increased in human AKI came from a case report by Leaf et al95 in 2010. The report described a 45-year-old man who presented with rhabdomyolysis-induced AKI, and was found to have low circulating levels of 1,25D and high levels of PTH. Plasma cFGF23 levels were increased at 619 RU/mL (reference range, ≤180 RU/ mL). The investigators proposed that FGF23-mediated inhibition of CYP27B1 in the setting of AKI, as in CKD, could explain the finding of reduced 1,25D levels despite increased PTH. Alternatively, the reduced 1,25D levels could have been caused by the reduced availability of 25-hydroxyvitamin D substrate and/or reduced CYP27B1 activity resulting from AKI alone. Subsequent studies confirmed the initial observation of increased FGF23 in human AKI, including studies performed in infants, children, and adults across a variety of AKI settings (Table 2). Zhang et al96 measured plasma cFGF23 levels in 20 critically ill adults with AKI from various etiologies and reported that median cFGF23 levels were 1,948 RU/mL. Leaf et al97 measured plasma cFGF23 levels in 30 hospitalized patients with AKI and reported that median cFGF23 levels were 1,471 RU/mL, which were 5.6-fold higher than an age-matched group of hospitalized patients without AKI. Furthermore, higher cFGF23 levels correlated with lower 1,25D levels (Rs = -0.39), supporting the possibility of FGF23-mediated inhibition of CYP27B1 in AKI.97 cFGF23 Versus iFGF23 in AKI As discussed earlier, two main assays are used to measure circulating FGF23 levels: the intact FGF23 assay detects only the intact hormone and the C-terminal FGF23 assay measures both the intact hormone as well as C-terminal fragments; results from the latter are sometimes referred to as “total” FGF23.98 Most studies of FGF23 in human AKI used the C-terminal assay and reported cFGF23 (ie, total) levels exclusively (Table 2). Markedly increased circulating levels of FGF23 in AKI detected with the cFGF23 assay are consistent with increased FGF23 production (or impaired excretion), but they do not indicate the relative amounts of biologically active intact hormone (iFGF23) versus biologically inactive C-terminal fragments (cFGF23). Accordingly, studies that simultaneously measured FGF23 using both the intact and C-terminal assays are useful in assessing

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Table 2. Human studies of FGF23 and AKI Study

Patients

Setting

AKI definition

Sample type

FGF23 assay*

Findings

Prediction of AKI Ali et al,150 2013

19 children

CS

pRIFLE153

Plasma

cFGF23

Brown et al,151 2014

3,241 older adults

OP

ICD-9-CM

Plasma

cFGF23

Hanudel et al,100 2016

32 children

CS

"SCr ≥50%

Plasma

cFGF23 and iFGF23

Leaf et al,99 2016

250 adults

CS

KDIGO ≥ stage 1 (SCr only)

Plasma

cFGF23

Leaf et al,112 2017

350 adults

ICU

KDIGO ≥ stage 1 (SCr only)

Urine

cFGF23

Rygasiewicz et al,113 2018

79 adults

ICU

KDIGO ≥ stage 1

Plasma

cFGF23 and iFGF23

Volovelsky et al,111 2018

41 infants

CS

"SCr ≥100%

Serum

cFGF23

Median preoperative cFGF23 levels were 4.7-fold higher in patients who did versus did not develop postoperative AKI (323 and 69 RU/mL, respectively) Median peak postoperative cFGF23 levels were 5.2-fold higher in patients who did versus did not develop postoperative AKI (1,010 and 196 RU/mL, respectively) Patients in the highest versus lowest quartiles of cFGF23 had a 1.99fold (95% CI, 1.04-3.80) higher adjusted odds of hospitalization for AKI Median preoperative cFGF23 levels were 3.7-fold higher in patients who did versus did not develop postoperative AKI (437 and 119 RU/mL, respectively), whereas median preoperative levels of iFGF23, Ca, PO4, and PTH were similar between groups Preoperative SpO2 correlated inversely with cFGF23 and iFGF23 Postoperatively, cFGF23 and iFGF23 increased by 2 h Longitudinal iFGF23 (but not cFGF23) levels were higher in AKI versus non-AKI patients Median preoperative cFGF23 levels were similar between patients who did versus did not develop postoperative AKI Median cFGF23 levels at end-CPB were 2.2-fold higher in patients who did versus did not develop postoperative AKI (657 and 298 RU/mL, respectively), and on POD1 were 3.3-fold higher in patients who did versus did not develop postoperative AKI (1,434 and 432 RU/mL, respectively) Patients in the highest versus lowest quartile of urinary cFGF23 had a 3.9-fold (95% CI, 1.6-9.5) higher adjusted odds of AKI/death Plasma levels of Ca, iCa, PTH, 25D, 1,25D, and DBP were not associated with AKI/death, and a trend (P = .05) was observed for PO4 and AKI/death Median cFGF23 (but not iFGF23) levels on arrival to the ICU were 4.2-fold higher in patients who did versus did not develop AKI (158 and 38 RU/mL, respectively) Median cFGF23 levels obtained 4-8 h postoperatively were 1.6-fold higher in patients who did versus did not develop AKI, with a trend (P = .07) toward significance

ICU

AKIN ≥ stage 1 (SCr only)

Plasma

cFGF23

Prediction of outcomes in established AKI 20 adults (12 with Zhang et al,96 2011 AKI and 8 controls) 60 adults (30 with AKI and 30 controls)

HP

AKIN ≥ stage 1 (SCr only)

Plasma

cFGF23

Leaf et al, 2013152

Same cohort as above

HP

AKIN ≥ stage 1 (SCr only)

Plasma

cFGF23

(continued on next page)

M. Christov et al.

Leaf et al,97 2012

Median cFGF23 levels were 7.7-fold higher in AKI versus control patients (1,948 and 252 RU/mL, respectively) Median cFGF23 levels were 8.2-fold higher in hospital nonsurvivors versus survivors (4,446 and 544 RU/mL, respectively) Median cFGF23 levels were 5.6-fold higher in AKI versus controls patients (1,471 and 263 RU/mL, respectively) In patients with AKI, higher cFGF23 levels associated with a higher risk of RRT/death, including after adjustment for age and SCr (OR, 13.7; 95% CI, 1.8-108) Higher cFGF23 levels were associated cross-sectionally with greater severity of sepsis

63

relative amounts of FGF23 production and cleavage because cleavage will reduce circulating levels of iFGF23 but will not affect cFGF23 levels. Leaf et al99 measured preoperative and postoperative plasma cFGF23 levels longitudinally in 250 adult patients undergoing cardiac surgery. In a subcohort of 18 patients who developed severe AKI and 18 age- and baseline estimated glomerular filtration rate (eGFR) −matched patients who did not develop AKI, they also measured plasma iFGF23 levels longitudinally. They found that although cFGF23 levels were increased markedly (»100-fold) postoperatively in patients who did versus did not develop severe AKI, iFGF23 levels in comparison were only mildly (»2-fold) higher postoperatively in AKI versus non-AKI patients. Similar findings were reported by Hanudel et al in a cohort of 32 children undergoing cardiac surgery.100 These data are consistent with marked increases in both the production and cleavage of FGF23 in AKI. Similar findings have been reported in mice exposed to acute inflammatory stimuli, and in animal and human studies of iron-deficiency anemia.22 Mechanisms of FGF23 Increase in AKI

*In all cases, FGF23 ELISAs from Immutopics/Quidel (San Diego, CA) were used.

Abbreviations: 25D, 25-hydroxyvitamin D; AKI/death, composite outcome of incident acute kidney injury or in-hospital mortality; AKIN, Acute Kidney Injury Network; CI, confidence interval; CPB, cardiopulmonary bypass; CS, cardiac surgery; HP, hospitalized patients; iCa, ionized calcium; ICD-9-CM, International Classification of Disease, 9th revision, Clinical Modification; DBP, vitamin D-binding protein; KDIGO, Kidney Disease: Improving Global Outcomes; OP, outpatient; OR, odds ratio; POD, postoperative day; pRIFLE, pediatric-modified Risk, Injury, Failure, Loss, End-stage kidney disease; RRT/death, composite of need for RRT or in-hospital mortality; SpO2, blood oxygen saturation.

Patients in the highest versus lowest quartiles of cFGF23 and iFGF23 had a 3.84-fold (95% CI, 2.31-6.41) and 2.08-fold (95% CI, 1.03-4.21) higher adjusted odds of 60-day mortality Plasma/serum levels of PTH, 25D, 1,25D, Ca, and PO4 were not associated with mortality Patients in the highest versus lowest quartiles of cFGF23 and iFGF23 had a 3.52-fold (95% CI, 1.96 to 6.33) and 1.93-fold (95% CI, 1.12 to 3.33) higher adjusted odds of 60-day mortality cFGF23 and iFGF23 cFGF23 and iFGF23 817 adults with AKI requiring RRT 710 adults with and without AKI Leaf et al,114 2018

ICU ICU

Requirement for RRT KDIGO ≥ stage 1 (SCr only)

Plasma Plasma

Findings Patients Study

Table 2 (Continued)

Setting

AKI definition

Sample type

FGF23 assay*

FGF23 and klotho

Preclinical studies corroborated the earlier-described findings in human beings and showed that circulating FGF23 levels increase in several animal models of AKI. Christov et al101 showed in a folic acid nephropathy mouse model that plasma FGF23 levels are increased in mice with AKI as early as 4 to 6 hours after induction of renal injury. Both intact and C-terminal FGF23 levels were increased, the latter as high as 20- to 40-fold over baseline, reaching levels similar to those reported in mice with CKD.102 Increased plasma FGF23 levels subsequently have been reported in mice or rats with AKI owing to other etiologies, including sepsis and hemorrhage,103 nephrectomy,104 and urinary obstruction.41 Classic regulators of FGF23 production, such as PTH, 1,25D, and phosphate, did not appear to play a significant role in this acute increase of FGF23 in AKI because the increase in FGF23 in the folic acid nephropathy model occurred even in mice in which PTH or the vitamin D receptor was genetically deleted.101 Furthermore, a low-phosphate diet did not prevent the FGF23 increase in this AKI model, suggesting that additional mechanisms are at play (Fig. 4). Similarly, a study of bilaterally nephrectomized rats found that prior parathyroidectomy did not affect the increase in plasma FGF23 levels in that model of AKI.104 Given that the kidneys play at least a modest role in FGF23 clearance, impaired excretion or catabolism of FGF23 by the kidneys in the setting of AKI theoretically could contribute to increased circulating levels (Fig. 4). Supporting this notion, in the folic acid nephropathy

M. Christov et al.

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Inflammation

↑FGF23 production by osteocytes

↑FGF23 production by extraskeletal tissues (e.g., kidney, spleen, bone marrow)

FGF23

Reduced renal clearance Figure 4. Potential mechanisms of increased FGF23 in AKI. Circulating levels of FGF23 are increased in both animals and humans with AKI. Multiple sources may contribute to increased circulating levels of FGF23 in AKI, including inflammation, which up-regulates FGF23 production in both osteocytes and in extraskeletal tissues, as well as reduced renal clearance. FGF23, in turn, also may increase inflammation, resulting in a positive feedback loop. Pathways that are less well established are shown in dotted lines.

model in mice the half-life of exogenously administered FGF23 was prolonged by 50% in AKI.101 In addition, Mace et al104 found that circulating FGF23 levels increased by a factor of 2.4 within 15 minutes after bilateral nephrectomy in rats. Interestingly, circulating FGF23 levels remained stable after the initial increase. Thus, impaired renal clearance may be only a modest and transient contributor to the increased circulating levels of FGF23 observed in AKI, with other factors likely contributing as well (Fig. 4).104 Increased production of FGF23 in bone is likely the main contributor to increased circulating levels of FGF23 in AKI. In a folic acid nephropathy mouse model, increased Fgf23 mRNA expression in bone was detected as early as 6 hours after AKI.101,105 This increase in bone Fgf23 mRNA expression, as well as the increase in circulating levels of FGF23, could be inhibited by pretreatment with the FGFR1 inhibitor PD173974.105 FGFR1 was implicated previously in the regulation of FGF23 production in bone, possibly as a receptor for locally produced canonical FGFs.106,107 Animal models of AKI have shown increased production of FGF23 in other organs, such as the injured kidneys, although the degree of contribution of renalderived FGF23 to circulating levels is unknown. In a unilateral ureteral obstruction (UUO) mouse model, Smith et al35 showed that Fgf23 mRNA is increased in renal tissues, although circulating intact FGF23 levels did not increase, and there was no effect on markers of mineral

metabolism. In their model, there was a transient increase in circulating cFGF23 levels within 24 hours after UUO. Mace et al,41 using a similar UUO model but in the rat, also showed increased Fgf23 mRNA expression in the obstructed kidneys. In that study, circulating iFGF23 levels increased and remained increased for up to 10 days after UUO. Mace et al41 further showed that removal of the 5/6 nephrectomy remnant in a CKD model, or removal of the obstructed kidney in a UUO model, did not attenuate the increase in circulating iFGF23 levels, suggesting that the production of FGF23 by the kidneys does not measurably affect circulating levels of FGF23. Recently, FGF23 production was found in the bone marrow, under the regulation of the erythropoietin system. Toro et al103 found that in mice with hemorrhagic shock−induced AKI or sepsis-induced AKI, bone marrow production of FGF23 significantly contributed to the increased circulating levels. In these animal models of AKI, inhibition of the erythropoietin receptor blocked the increase of Fgf23 mRNA expression in the bone marrow in AKI and attenuated the increase in circulating levels. The spleen was shown to produce FGF23 and contribute to increased circulating cFGF23 levels in a mouse model of acute inflammation using lipopolysaccharide.27 Given that AKI is a state of acute inflammation, it is possible that other tissues, such as the spleen, or other cell types, such as immune cells, can produce FGF23.

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FGF23 and klotho

Clinical Relevance of Increased FGF23 in AKI

FGF23 levels are associated with worse short-term outcomes, as discussed in the following section.

In the obstructed kidney model, Smith et al108 showed that locally produced FGF23 can augment myofibroblast activation and fibrogenesis via activation of transforming growth factor b−related pathways. Interestingly, when fibroblasts derived from normal kidneys were treated with FGF23 in vitro there was no effect on profibrotic programs, whereas fibroblasts primed by injury responded to FGF23 signaling, which was independent of klotho.35 Thus, there are some data that locally produced FGF23 by injured kidneys can feed forward and potentiate renal injury (Fig. 5). This observation is consistent with epidemiologic studies in patients with both chronic and acute forms of kidney injury. Specifically in CKD, higher plasma FGF23 levels are predictive of CKD progression,109,110 whereas in AKI higher plasma

FGF23 Is an Early Prognostic Marker of Incident AKI in Humans Cardiac surgery−associated AKI

Increased circulating levels of FGF23 have been reported to be an early marker of incident AKI (Table 2). In a cohort study of 250 adult patients undergoing cardiac surgery, Leaf et al99 found that plasma cFGF23 levels were increased significantly very early on—at the end of cardiopulmonary bypass—in patients who did versus did not develop AKI postoperatively. The early increase in plasma cFGF23 predated changes in other mineral metabolites, including calcium, phosphate, PTH, and

A Renal fibrosis

Increased inflammation

FGF23

Immunosuppression Endothelial dysfunction

Suppression of hematopoiesis

B Decreased autophagy Increased cell senescence and apoptosis

Renal fibrosis

Klotho

Vascular calcification

Pathologic cardiac remodeling Endothelial dysfunction

Figure 5. Postulated renal and extrarenal effects of (A) FGF23 and (B) klotho in AKI.

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vitamin D metabolites, suggesting alternative mechanisms and stimuli driving FGF23 production (Figs. 1 and 4). Interestingly, the area under the receiver operating characteristic curve, a measure of the predictive performance of an exposure, was higher for plasma cFGF23 (0.78) at the end of cardiopulmonary bypass compared with several urinary injury biomarkers, including neutrophil gelatinase-associated lipocalin (0.61), kidney injury molecule-1 (0.65), and n-acetyl-b-(D)-glucosaminidase (0.60).99 Early increase of circulating FGF23 in patients with AKI after cardiac surgery also has been reported by other investigators, including in infants111 and in children100 (Table 2). Intensive care unit−associated AKI

Increased circulating levels of FGF23 also have been reported to be an early and independent prognostic marker of incident AKI among critically ill patients (Table 2).112,113 Furthermore, in a prospective cohort study of 350 critically ill adult patients, Leaf et al112 recently reported that higher cFGF23 levels measured in the urine within 48 hours of admission to the intensive care unit (ICU) were associated independently with incident AKI or death. Additional research is needed to determine whether urinary FGF23 could be a useful prognostic test for AKI and other kidney-related outcomes in settings in which obtaining plasma may not be feasible.

FGF23 Is a Prognostic Marker of Adverse Outcomes in Patients With Established AKI In addition to its potential role as an early marker of incident AKI, FGF23 also may have a role as a prognostic marker of adverse outcomes among patients with established AKI (Table 2). Leaf et al114 recently measured plasma cFGF23 and iFGF23 in two large cohorts of critically ill adults: patients with AKI requiring renal replacement therapy (RRT) who enrolled in the Acute renal failure Trial Network study (n = 817), and a general cohort of critically ill patients with and without AKI who enrolled in the Validating Acute Lung Injury biomarkers for Diagnosis study (n = 710). In both cohorts, patients in the highest versus lowest quartiles of cFGF23 and iFGF23 had a significantly increased risk of 60-day mortality after multivariable adjustment.114 Whether higher levels of FGF23 contributed directly to adverse outcomes in AKI, as they do in CKD, either through classic or off-target effects, is an intriguing possibility that requires further study. Off-target effects of FGF23 that have been reported include profibrotic effects on the kidneys,35 impairment of immune function,47,115 impairment of erythropoiesis,46 and toxic effects on the endothelium116 and cardiovascular system (Fig. 5).16

DECREASED KLOTHO IN AKI Klotho Is Decreased in Experimental AKI Sugiura et al117 described decreased klotho expression in experimental AKI. After bilateral ischemia-reperfusion injury (IRI) in rats, lower renal klotho mRNA and protein levels were observed. Hu et al118 further showed in murine models of bilateral IRI that renal klotho mRNA expression and protein levels, as well as urinary and plasma klotho levels, were decreased after IRI. These changes were detected as early as 3 hours after IRI.118 Notably, renal klotho mRNA expression and plasma klotho levels changed in parallel, preceded changes in plasma creatinine and neutrophil gelatinase-associated lipocalin, and were restored within 7 to 10 days after IRI.

Klotho Is Decreased in Human AKI Current data on klotho in human AKI are insufficient to draw firm conclusions (Table 3). Hu et al118 examined urinary klotho levels in 17 patients with established AKI from heterogeneous causes and showed that levels were significantly lower in these patients than in 14 healthy volunteers (means § SD, 4.85 § 1.69 versus 25.38 § 4.08 fmol per mg of creatinine; P <.01). The control group, however, had lower baseline serum creatinine (SCr) levels, among other important differences. In a different study, Kim et al119 showed that urinary klotho levels were significantly lower in patients with prerenal versus intrinsic AKI, and postulated that assessment of urinary klotho levels might help differentiate these two entities. More recently, Seo et al120 examined kidney biopsy samples from patients with AKI from acute tubular necrosis or acute tubulointerstitial nephritis. They classified patients into three categories according to their renal klotho protein levels (low, medium, or high) and found that patients in the low klotho group had higher peak SCr values and required RRT more frequently compared with patients in the medium or high klotho groups. In a study in kidney transplant recipients, Castellano et al121 studied renal klotho protein levels in cadaveric kidneys, and found that the levels decreased dramatically after transplantation in those with delayed graft function. In addition, patients who experienced delayed graft function were found to have significantly lower serum klotho levels at 2 years after transplantation when compared with patients who did not have delayed graft function after transplantation. Other studies have reported contradictory findings with respect to soluble klotho levels in human AKI. Seibert et al122 found that serum klotho levels were higher in patients with AKI (assessed at the time of renal consultation) compared with a nonmatched outpatient control group without AKI (567.6 § 294.4 versus 403.5 §

Study

Patients

Associations with established AKI 31 adults (17 AKI, Hu et al,118 2010 14 HV)

Setting

AKI definition

Sample type

Klotho assay*

Inpatient renal consult

"SCr ≥50% or ≥2 mg/dL increase Biopsy-proven

Urine

Immunoblot Results: mean urine klotho/Cr levels were significantly lower in AKI versus HV (4.85 § 1.69 versus 25.38 § 4.08 fmol/mg of Cr, P < .01) Comments: baseline SCr was higher in AKI patients versus HV Rabbit anti- Results: patients were stratified according to three tiers of renal klotho human expression (low, medium, or high) polyclonal Peak SCr was higher and RRT was more frequent in the low klotho group antibody versus the medium or high groups Renal klotho protein levels correlated negatively with peak SCr Comments: patients with evidence of CKD on kidney biopsy were excluded IBL ELISA Results: patients with prerenal and intrinsic AKI had similar SCr levels Urine klotho/Cr levels were lower in prerenal versus intrinsic AKI (means § SD, 174 § 292 versus 381 § 630 ng/g of Cr; P = .001) There was no difference between groups in serum klotho levels IBL ELISA Results: renal klotho protein expression was decreased dramatically in cadaveric kidneys from patients with DGF Patients with versus without DGF also had lower levels of serum klotho at »2 years after transplantation Immunoblot Results: urine klotho/Cr levels assessed at a single time point (at time of AKI (KM2076) diagnosis [cases] or within 24 h of ICU admission [controls]) were significantly lower in AKI versus controls: median (IQR), 7 (5-10) versus 28 (1159) fmol/mg of Cr; P < .001) Comments: ICU controls were matched by age, sex, and baseline eGFR IBL ELISA Results: Serum klotho levels were higher in AKI (assessed at the time of renal consultation) versus control patients (means § SD, 567.6 § 294.4 versus 403.5 § 152.5 pg/mL; P < .01) Comments: AKI and controls were not matched by age or baseline eGFR Controls had normal kidney function and absence of proteinuria or HTN

Seo et al,120 2015

21 adults with ATN Inpatients or ATIN on kidney biopsy

Kim et al,119 2016

61 adults (42 prerenal, 19 intrinsic AKI)

ICU or ward

AKIN ≥ stage 1

Serum and urine

Castellano et al,121 2016

30 adults (15 developed DGF)

Kidney transplant

DGF

Serum

Neyra et al,123 2016 106 adults (54 AKI, 52 ICU controls)

ICU

KDIGO ≥stage 2 Urine

Seibert et al,122 2017

46 adults (30 AKI, 16 controls)

Inpatients

AKIN ≥ stage 1

Serum

Prediction of AKI Liu et al,140 2015

35 adults (19 AKI)

CS

AKIN ≥ stage 1 (SCr only)

Serum

ELISA

60 adults (30 AKI)

CS or CA

"SCr ≥50%

Urine

IBL and SSBT ELISA

Torregrosa et al,141 2015

Tissue

Findings

FGF23 and klotho

Table 3. Human Studies of Klotho and AKI

Results: serum klotho levels were lower in AKI versus non-AKI patients both immediately after CS and 4 h after CS: means § SD, 102 § 17 versus 124 § 21 U/L, P < .01 and 103 § 14 versus 103 § 14 U/L, P = .01 However, klotho levels increased as early as day 1 after CS such that levels were no longer different in AKI versus non-AKI patients Results: urine samples were collected 12 h after CS or after CA (single time point only) Urine klotho/Cr was not different in patients with AKI versus no AKI (means § SD, 2.45 § 0.26 versus 2.04 § 0.20 ng/mg of Cr, P = NS by SSBT and 1.60 § 0.30 versus 1.24 § 0.30, P = NS by IBL) Comments: AKI patients had lower baseline eGFR than those without AKI (60 § 18 versus 78 § 15; P < .01). No correlation between the 2 klotho assays was found

Abbreviations: AKIN, Acute Kidney Injury Network; ATIN, acute tubulointerstitial necrosis; ATN, acute tubular necrosis; CA, coronary angiography; CS, cardiac surgery; DGF, delayed graft function, defined as need for RRT within 7 days after transplantation; HTN, hypertension; HV, healthy volunteer; IBL, Immuno-Biological Laboratories Co, Ltd; IQR, interquartile range; KDIGO, Kidney Disease: Improving Global Outcomes; SSBT, Shangai Sunred Biological Technology Co, Ltd.

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152.5 pg/mL; P <.01). However, Neyra et al123 reported that urinary klotho levels normalized to the urinary creatinine (Cr) concentration were significantly lower in AKI patients at the time of diagnosis versus ICU controls without AKI: median of 7 (interquartile range, 5-10) versus 28 (interquartile range, 11-59) fmol/mg of Cr (P < .001). It is important to note that the former study involved cases with AKI stage ≥1 (Acute Kidney Injury Network criteria)124 and nonmatched controls from the outpatient setting, whereas the later study included cases with AKI stage ≥2 (Kidney Disease: Improving Global Outcomes criteria)125 and ICU-matched controls by age, sex, and baseline eGFR (Table 3). In addition, the former study measured soluble klotho levels in the serum, whereas the latter measured it in the urine. Finally, different klotho assays were used in these studies (discussed further in the following section). Measurement of Soluble Klotho Levels Lack of a reliable, high-throughput assay for measurement of soluble klotho levels in human blood and urine samples has been a major challenge in performing largescale studies in different clinical settings, such as AKI.64 A commonly used commercial enzyme-linked immunosorbent assay (ELISA) kit by Immuno-Biological Laboratories (Minneapolis, MN) yielded different results in fresh versus stored serum samples, and klotho levels measured by ELISA were less stable after repeated freeze-thaw cycles when compared with the immunoprecipitation-immunoblot assay (immunoprecipitation with sb106 anti-klotho antibody and immunoblot with rat anti-human klotho monoclonal antibody KM2076; Trans Genic, Inc, Kobe, Japan).126 Furthermore, klotho was shown to be highly unstable in human urine even when stored at -80˚C, as measured with the Immuno-Biological Laboratories ELISA kit.127 Although decreased levels of both renal tissue klotho and soluble klotho have been associated with adverse outcomes in clinical and preclinical studies, it is currently unclear which form of klotho (eg, soluble versus membrane-bound in the tissue) is most biologically relevant, and therefore associations drawn between soluble klotho levels and clinical outcomes should be interpreted with caution. Finally, it is possible that available klotho assays are not able to differentiate between intact soluble klotho, klotho fragments, and klotho complexed with other proteins (including FGF23). Potential Mechanisms of Klotho Deficiency in AKI AKI is a state of intense inflammation, resulting in release and activation of both local and systemic mediators. Inflammatory cytokines such as tumor necrosis factor-a and tumor necrosis factor−like weak inducer of apoptosis have been shown to down-regulate renal

M. Christov et al.

klotho mRNA and protein expression through nuclear factor-kB activation in a folic acid nephropathy mouse model of AKI.128 In a different experiment, tumor necrosis factor-a and interferon-g reduced renal klotho protein and mRNA expression in mouse models of inflammatory bowel disease.129 The reduced renal klotho expression was postulated to be mediated by an increased expression of inducible nitric oxide synthetase and nitric oxide production, based on in vitro experiments. Importantly, the precise mechanisms of how this inflammatory milieu down-regulates klotho expression has not been fully elucidated.129 Similarly, oxidative stress induced by hydrogen peroxide also has been shown to decrease klotho mRNA and protein expression in vitro in a mouse kidney cell line.130 More recently, other potential mechanisms of klotho down-regulation have been postulated, and include differential transcriptional splicing, resulting in decay of klotho mRNA.80 In CKD, mechanisms of decreased klotho expression include hyperphosphatemia91 and hypermethylation,131,132 or deacetylation of the klotho gene promoter by inflammatory cytokines or uremic toxins such as indoxyl sulfate. Thus far, it is unclear which of these mechanisms, if any, are responsible for the decreased expression of klotho that occurs in AKI. Potential Clinical Relevance of Low Klotho in AKI Given the limitations of the existing assays for measurement of soluble klotho (discussed earlier), no large-scale human studies have conclusively examined the association between circulating levels of soluble klotho and clinical outcomes in AKI. The following section highlights data from several animal studies and the limited data available from two small human studies that assessed the potential role of low klotho in AKI. Klotho is associated with severity of kidney insult and subsequent renal fibrosis

Acute decreases in renal klotho mRNA expression and protein levels in AKI have been shown in several murine models, including UUO,60 cisplatin nephrotoxicity,81 sepsis, IRI,118 and post-IRI CKD.63 In the latter model, animals were treated with a high-phosphate diet starting 2 weeks after IRI, and CKD was detected (both functionally and histologically) 20 weeks later. Importantly, renal klotho mRNA and protein expression recovered 7 to 10 days after IRI, but then showed a subacute, progressive, and irreversible decrease starting at day 14 after IRI, particularly in mice exposed to prolonged ischemia. Furthermore, klotho-deficient (heterozygous knockout) mice showed more severe kidney fibrosis and greater progression to CKD when compared with wild-type or transgenic mice that overexpressed klotho. Similarly, administration of exogenous recombinant klotho protein administered daily for 4 consecutive days beginning

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FGF23 and klotho

1 day after IRI accelerated kidney recovery, reduced kidney fibrosis, enhanced endogenous renal klotho mRNA and protein expression, and attenuated the occurrence of CKD after IRI.63 Administration of exogenous klotho protein after AKI ameliorates subsequent CKD and pathologic cardiac remodeling

Hu et al87 found that administration of exogenous recombinant klotho protein (on a daily basis for 4 consecutive days, beginning 1 day after renal insult) attenuated pathologic cardiac remodeling 20 weeks after IRI-induced AKI. In addition, by using a CKD model of unilateral nephrectomy plus contralateral IRI, they showed that the chronic administration of recombinant klotho protein slowed CKD progression and protected the heart from cardiac fibrosis and hypertrophy. In this CKD model, administration of klotho decreased plasma phosphate and FGF23 levels when compared with vehicle administration. Plasma levels of soluble klotho paralleled renal klotho mRNA and protein levels. The investigators also observed that exogenous klotho therapy seemed to restore endogenous renal klotho expression. Klotho therapy also may have a role in nephrotoxic AKI. In an experimental model of cisplatin-induced AKI, klotho administration down-regulated the expression of organic cation transporter-2, thereby reducing the uptake of cisplatin into tubular cells and attenuating renal injury.81 Whether up-regulation of endogenous renal klotho, soluble klotho, or both could play an important role in kidney protection is unknown. In addition, understanding the specific mechanisms of how exogenous klotho confers kidney protection in AKI, and the precise molecular targets of klotho, constitute areas of intensive research without clear answers. Experimental data have suggested that klotho can protect the kidney by multiple putative mechanisms: suppression of apoptosis81,133 and cell senescence,134,135 antifibrosis,60,61,136-138 and up-regulation of autophagy62,63 in kidney tubular cells (Fig. 5). Similarly, soluble klotho has been reported to ameliorate albuminuria by suppressing transient receptor potential canonical 6 activity in podocytes and protecting the glomerular filtration barrier.139 Klotho as a prognostic marker of incident AKI

Only two studies to date have studied the ability of soluble klotho to predict incident AKI. Both studies were conducted in the cardiac surgery perioperative setting (Table 3). Liu et al140 examined 35 adult patients undergoing cardiac surgery, of whom 19 (54%) developed postoperative AKI. They found no differences in age, baseline eGFR, or preoperative serum klotho levels between patients who did or did not develop

postoperative AKI. However, serum klotho levels obtained immediately (0 h) and 4 hours after cardiac surgery were each significantly lower in patients with versus without AKI, but subsequently the levels rapidly increased in patients with AKI such that they were no longer different between groups by postoperative day 1. The area under the receiver operating characteristic curve for the early prediction of AKI was 0.81 (95% confidence interval, 0.66-0.95) for serum klotho measured immediately after cardiac surgery and 0.75 (95% confidence interval, 0.59-0.92) for serum klotho measured 4 hours after cardiac surgery. A subsequent study by Torregrosa et al141 was unable to replicate these results. In a cohort of 60 adult patients undergoing cardiac surgery or coronary angiography (30 of whom developed postoperative or postprocedure AKI), urinary klotho/Cr levels were no different in patients with versus without AKI when measured 12 hours after cardiac surgery or coronary angiography. It is important to emphasize that klotho was measured in different types of biological samples (serum versus urine) and at different time points, and that different commercially available ELISAs were used in these studies (Table 3). In summary, the measurement of serum or urinary klotho levels in human beings is inconsistent and there is no consensus on which assay to use. Thus, currently available data on soluble klotho levels in human AKI should be interpreted with caution.

FGF23 AND KLOTHO AS THERAPEUTIC TARGETS IN AKI Overview Currently, no specific strategies are clinically available to modify the changes in FGF23 and klotho observed in AKI (Table 1 and Fig. 3). However, data from animal and human studies allow us to contemplate potential therapeutic approaches that one day may become available to prevent, attenuate, or treat AKI (Fig. 6). Modifying the Increase in FGF23 or its Downstream Effects Given the myriad off-target adverse effects of excessively high circulating FGF23 levels, neutralization of FGF23 might appear to be a promising therapeutic strategy to improve outcomes in AKI (Fig. 6). However, such a strategy likely would have undesired consequences, namely worsening of hyperphosphatemia, especially in patients with nonoliguric AKI who do not require RRT. This was shown in a 5/6 nephrectomy CKD model in rats, in which administration of an FGF23 neutralizing antibody resulted in improved PTH and calcium levels, but resulted in hyperphosphatemia, increased aortic calcification, and increased mortality.142 Similarly, the use

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vivo.33 Interestingly, the effect of administration of cFGF23 on decreased urinary phosphate excretion was confirmed in a separate study performed in mice using a 5/6 nephrectomy CKD model, however, the treated group also had lower serum phosphate levels compared with vehicle-treated CKD mice for unclear reasons.34 An additional site of interference of FGF23 downstream signaling that could be targeted therapeutically was FGFR4, which has been implicated in some of the off-target effects of FGF23 in CKD, including left ventricular hypertrophy and increased production of inflammatory mediators.15,90 This could be accomplished by using a specific FGFR4 inhibitor.145 An advantage of this approach would be to interfere with some of the off-target (presumably adverse) effects of FGF23 while preserving the physiologic roles of FGF23 in maintaining phosphate and vitamin D homeostasis, which are mediated by FGFR1.

FGF23-based Therapeutics • • •

FGF23 neutralizing synthetic antibodies C-terminal FGF23 Downstream signaling inhibition (eg, FGFR4 inhibitorF)

FGF23

High FGF23 +

+

+

+

1,25D

Phosphate

-

+

-

Low Klotho

Klotho-based Therapeutics • • • •

Klotho

Exogenous administration of recombinant Klotho protein Viral delivery of Klotho cDNA Epigenetic increase of endogenous Klotho (eg, via demethylation or deacetylation of the Klotho gene promoter) Pharmacological increase of endogenous Klotho (eg, RAAS blockers, PPAR-γ agonists, statins)

Modifying the Decrease in Klotho Expression

Figure 6. FGF23 and klotho-based therapeutics in AKI. The diagram represents FGF23-based therapies (top) and their potential interactions with key mineral metabolism markers in AKI (green arrows) and klotho-based therapies (bottom) and their potential interactions with key mineral metabolism markers in AKI (brown arrows). Gray arrows represent known interactions between mineral metabolism markers in CKD models. Abbreviations: cDNA, complementary DNA; PPAR-g agonist, peroxisome proliferator-activated receptor g; RAAS, reninangiotensin-aldosterone system.

of a modified anti-FGF23 antibody (burosumab) in humans with X-linked hypophosphatemic rickets, a condition caused by increased circulating FGF23 levels, has also resulted in increases in serum phosphate and 1,25D levels (in this case the desired effect).143,144 Thus, based on the available evidence, the only viable way in which an anti-FGF23 antibody such as burosumab could be studied in patients with AKI would be in those with severe oliguric AKI requiring RRT, whereby hyperphosphatemia would be avoided through extracorporeal clearance. An alternative approach would be to interfere with FGF23 signaling, either at the site of binding to its receptor or by inhibiting downstream signaling cascades. The isolated C-terminal fragment of FGF23 was shown to interfere with binding of FGF23 to FGFR1 in vitro and, when injected in supraphysiological concentrations, to interfere with the phosphaturic action of FGF23 in rats in

Renal and soluble klotho levels decrease in both animal and human models of AKI, and klotho has many important pleiotropic actions such as inhibition of apoptosis81,133 and cell senescence,134,135 antifibrosis,60,61,136-138 and up-regulation of autophagy62,63 in kidney tubular cells. Thus, counteracting the decrease in klotho expression that occurs in AKI represents a promising strategy to prevent/ameliorate AKI and related outcomes. Several animal studies have explored this strategy. Specifically, one strategy is to use viral delivery of klotho complementary DNA, which was shown to reduce apoptosis in a bilateral IRI rat model of AKI.117 Alternatively, administration of exogenous recombinant klotho protein might restore the soluble levels of this hormone and also potentially could promote endogenous production of renal klotho. In a murine IRI model, Hu et al118 injected the soluble extracellular domain of klotho intraperitoneally 30 or 60 minutes after ischemia. This treatment led to an attenuated increase in SCr after injury and also improved renal histology compared with vehicle-treated animals. Furthermore, the same group of investigators found that repeated administration of soluble klotho starting 1 day after IRI led to reduced fibrosis and improved recovery from AKI.63 An alternative approach would be to increase endogenous production of klotho by overcoming the mechanisms that lead to decreased expression of the protein in

Table 4. Potential Applications of Klotho in AKI Biomarker Diagnostic Prognostic

 Early AKI recognition  AKI progression  AKI recovery  CKD, ESRD, or CVD after AKI

Therapeutic agent Prophylactic Therapeutic

 Prevention of AKI in high-risk groups (eg, cardiac surgery, early sepsis)  Attenuate AKI progression  Promote AKI recovery  Attenuate the risk of CKD, ESRD, or CVD after AKI

FGF23 and klotho

AKI. Because the main source of soluble klotho is the kidney, in states of kidney injury there may be resistance to such strategies due to active injury and inflammation of the organ. However, it is worth noting that in a CKD model of partial renal ablation, treatment with the vitamin D−receptor agonist, paricalcitol, led to increased serum levels of soluble klotho and reduced serum phosphate and FGF23 levels compared with a control group. In this study, the source of increased soluble klotho levels could not be identified, and did not appear to originate from the kidneys.146 Experimental up-regulation of klotho expression by 1,25D also has been confirmed in other studies.147,148 Other agents that potentially can increase endogenous klotho expression are angiotensin II −receptor antagonists, peroxisome proliferator-activated receptor-g agonists, and statins (Figs. 1 and 6).67-70,149 Thus far, the effect of these agents on klotho has been shown only in preclinical studies, except for the use of valsartan, which was shown to increase plasma klotho levels in patients with diabetic nephropathy after 24 weeks of therapy.68 In summary, klotho has the potential not only to be a predictive or prognostic biomarker of AKI, but it is also a promising therapeutic agent that might attenuate damage, promote recovery, and ameliorate the risk of CKD or CVD after AKI (Table 4). Carefully designed, randomized controlled trials in humans are needed to test this hypothesis.

SUMMARY Increased circulating FGF23 levels and reduced klotho expression are found in both animal and human models of AKI. These changes are associated with more severe renal injury and less recovery of renal function in animal models, as well as increased morbidity and mortality in patients. Effective strategies to modify the levels or downstream effects of FGF23 and klotho overactivity and underactivity, respectively, are currently only speculative, but represent a unique therapeutic opportunity to improve the dismal outcomes associated with AKI.

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