Fibroblast Growth Factor (FGF) 23 and αKlotho in Acute Kidney Injury: Current Status in Diagnostic and Therapeutic Applications

Abstract

Fibroblast Growth Factor-(FGF) 23 and αKlotho are circulating mineral regulatory substances that also have a very diverse range of actions. Acute Kidney Injury (AKI) is a state of high FGF23 and low αKlotho. Clinical association data for FGF23 are strong but the basic pathobiology of FGF23 in AKI is rather sparse. Conversely, preclinical data supporting a pathogenic role of αKlotho in AKI is strong but the human data are still being generated. This pair of substances can potentially serve as diagnostic and prognostic biomarkers. FGF23 blockade and αKlotho restoration can have prophylactic and therapeutic utility in AKI. The literature to date is briefly reviewed in this article.

I. INTRODUCTION

Fibroblast growth factor (FGF) 23 was cloned as a candidate gene of autosomal dominant hypophosphatemic rickets, and a differentially expressed gene in tumor-induced osteomalacia, which ushered in a major hormone countering phosphate accumulation(1). Beyond phosphate balance, FGF23 has been shown or postulated to regulate calcium and sodium homeostasis, iron metabolism, erythropoiesis, and inflammation(2).

αKlotho was cloned when serendipitous disruption of its locus led to premature multi-organ failure(3). The list of cellular actions conferred by αKlotho encompasses anti-oxidation, anti-apoptosis, pro-autophagy, pro-stem cell, and anti-fibrosis. Essentially, broad-based maintenance of cell health(1).

Chronic kidney disease (CKD) is a state of FGF23 excess and αKlotho deficiency but their levels and roles in acute kidney injury (AKI) are less well defined. Many of the functions of FGF23 and αKlotho are highly relevant to the pathobiology of AKI. AKI is a systemic syndrome triggered by a sudden loss of kidney function, and brings forth significant morbidity, mortality, and burden on multiple organ systems(4, 5). Even when one evades the acute high mortality, survivors are at risk for CKD, end-stage kidney disease, and cardiovascular disease(6–9).

For a common serious syndrome with dire short- and long-term consequences, progress in diagnosis, prognosis, and therapy has been modest. We need means to diagnose AKI early, foresee the time course of recovery, and predict the risk of progression to CKD. For therapeutics, we need effective prophylaxis of AKI in high-risk situations, accelerate recovery in established AKI, prevent extra-renal complications, and forestall AKI-to-CKD transition. We will explore if FGF23 and αKlotho can fulfill these roles.

II. FGF23 and AKI

Multiple factors increase FGF23 production, including high blood pressure, phosphate loading, iron deficiency, hypoxia and inflammation(10). FGF23 actions are transduced by the FGF receptor 1 (FGFR1)(11, 12), and its co-receptor transmembrane αKlotho(13). FGF23 can also exerts action through binding to FGFR4 in the heart and liver, independently of αKlotho(14, 15). Metabolism of FGF23 includes cleavage of bioactive intact FGF23 (~30 kDa) into C-terminal (~12 kDa) and N-terminal (~18 kDa) fragments. FGF23 seems to be also cleared by the kidneys, possibly through filtration and/or catabolism(16). Circulating FGF23 includes the intact hormone (iFGF23) and the N- and C-termini. In states such as iron deficiency and inflammation, increased production of FGF23 is matched by FGF23 cleavage, with high levels of FGF23 fragments in circulation(17). Two assays used to measure circulating FGF23 levels are ‘C-terminal’ (cFGF23) which detects both C-terminal fragments and the full length peptide and ‘Intact’ (iFGF23) which measures only intact FGF23.

Elevated FGF23 levels have been observed in multiple studies of human AKI(10). Plasma cFGF23 levels were 5.6-fold higher in patients with AKI vs. age-matched patients without AKI(18). Note that increased FGF23 levels detected with the cFGF23 assay (intact + C-term) may reflect increased FGF23 production and/or impaired clearance but do not inform about FGF23 bioactivity. Few studies have used both cFGF23 and iFGF23 assays in conjunction. In adult patients undergoing cardiac surgery, plasma cFGF23 levels were markedly increased (~100-fold) postoperatively in patients who did vs. did not develop AKI, while iFGF23 levels were modestly (~2-fold) higher postoperatively in AKI vs. non-AKI patients(19). Similar findings were seen in a folic acid nephropathy mouse model(20). Therefore, both production and clearance of FGF23 may be affected in AKI.

In 250 adult patients undergoing cardiac surgery, plasma cFGF23 levels differentially increased at the end of cardiopulmonary bypass in patients with vs. without postoperative AKI, predating changes in other mineral metabolites.(19) Performance evaluation of utility of cFGF23 for early AKI diagnosis was modest (AUC=0.78) but superior to urinary kidney injury biomarkers(19). Similar observations were derived from cohorts of critically ill patients in which cFGF23 was measured in plasma and urine within 24-48 h of ICU admission(21, 22).

FGF23 levels may also have prognostic utility in AKI. In a large post-hoc analysis (n=1527 patients), increased risk of 60-day mortality was noted in patients with the highest vs. lowest quartiles of CFGF23 (~3.8 fold) and iFGF23 (~2.0 fold) (23).

The canonical mineral metabolism regulators of FGF23 production in bone seem not to play a major role in AKI(20, 24). Further, contribution of FGF23 production from other organs in acute injury/inflammation settings are not fully understood. FGF23 may augment myofibroblast activation and fibrosis via TGFβ-related pathways, opening an angle for therapeutic FGF23 blockade(25, 26). However, off-target effects of FGF23 such as impairment of immune(27, 28), endothelial(29), and cardiac function(15) are contenders and require further investigation. Therefore, it is still unclear if anti-FGF23 antibody (burosumab), C-terminal FGF23 (experimentally shown to interfere with FGF23 signaling at supra-physiologic concentrations) (30), or specific FGFR4 inhibition have clinical applications in attenuation of incident AKI or its consequences.

III. αKLOTHO IN AKI

Diagnosis.

αKlotho deficiency is universal in AKI animal models including ischemia-reperfusion injury (IRI)(31), unilateral ureteric obstruction(32) (UUO), sepsis induced by lipopolysaccharide (LPS) injection or cecal ligation and puncture (CLP)(33, 34), and nephrotoxins including cisplatin(35) (CP) and folic acid(36) (FA), indicating that αKlotho downregulation in the kidney is a general phenomenon after acute kidney insults (Table 1).

Table 1:

αKlotho levels in animal models of AKI

Animal model	Blood αKlotho	Urine αKlotho	Renal αKlotho assay	Kidney function	Kidney histology	Comments	Citation
Hypovolemia (phlebotomy 4 ml, Wistar rat	ND	ND	No change in mRNAwith Northern Blot from 30-120 min	ND	ND	Pre-renal insult did not change αKlotho mRNA within 120 minutes after phlebotomy (up to 15% blood volume/body weight). No information about Bp, renal function, and renal αKlotho protein.	(34)
LPS (10-20 mg/kg), mice	ND	ND	↓ Protein with IB and ↓ mRNA with qPCR at 3 hrs	↑ Scr and ↑ NGAL at 24 hrs		Dose dependent decrease of αKlotho in the kidney	(34, 46)
Cecal ligation and puncture (CLP), mice	ND	ND	↓ Protein with IB	↑ BUN ↓ Urinary volume	↑ Renal tubular damage scores	CLP caused lower survival, impaired renal function, impaired hepatic function, greater oxidative stress, inflammatory pathways (at the systemic and kidney tissue levels) in Kl/+ mice than WT mice	(33)
IRI (60 min), Wistar rat	ND	ND	↓ mRNA with qPCR ↓ protein with IB	↑ Scr	ATN	1. αKlotho mRNA ↓ at 6 hrs after IRI 2. Sort↑ at 6 hrs after IRI 3. αKlotho protein started to rise at 3 days and back to normal at 10 days	(37)
IRI (30 min), mice	↓ with IB-IP	↓ with IB	↓ with IB	↑ Scr	ATN	1. kl/+ mice with lower αKlotho had severer kidney damage and Tg-kl mice with higher αKlotho had milder kidney damage 2. αKlotho decline started 3 hours after IRI and early than elevation of NGAL	(40)
IRI (20-35 min), mice	ND	ND	↓ mRNA with qPCR ↓ protein with IB	↓ CICr ↑ ACR	Renal fibrosis at 20 weeks	1. Longer ischemia reduced more renal αKlotho 2. ↓ renal αKlotho is associated with ↑ renal fibrosis 3. ↑ dietary Pi causes ↓ renal αKlotho	(40)
Cisplatin (10 mg/Kg) once, mice	ND	ND	↓ mRNA with qPCR ↓ protein with IB Starting one day and stayed at low levels for one week	↑ Scr and ↑ACR	1. acute phase: ATN 2. chronic phase: renal fibrosis	1. Old mice had more fibrosis and higher SCr with less renal αKlotho 2. kl/+ mice had worse renal function and severer AKI and CKD 3. Tg-kl mice had better renal function and less AKI and less CKD	(35, 36, 47)
Folic acid, IP, (250 mg/kg) once, mice	ND	ND	↓ mRNA with qPCR ↓ protein with IB at one day after folic acid	↑ Scr	↑AKI scores. ↑ macrophages in the kidney	1. ↓ αKlotho mRNA and protein expression with more AKI pathological scores and macrophages in the kidney at day one 2. TWEAK and TNFa reduce renal αKlotho	(36)
UUO, mice	ND	ND	↓ protein with IB	ND	↑ renal fibrosis at up to 5 days	1. More fibrosis in kl/+ than WT mice 2. Lower renal αKlotho is associated with more renal fibrosis	(32)

ACR: albumin/creatinine ratio; AKI: acute kidney injury; ATN: acute tubular necrosis; Bp: blood pressure; BUN: blood urea nitrogen; CKD: chronic kidney disease; ClCr: creatinine clearance; eGFR: estimated glomerular filtration rate; hrs hours; IB: immunoblot; IRI: ischemia-reperfusion injury; kl/+: heterozygous αKlotho deficient mouse; LPS: lipopolysaccharide; min: minute; NGAL: neutrophil gelatinase-associated lipocalin; ND: not done; qPCR: quantitative PCR; Pi: phosphate; Scr: serum creatinine; Tg-kl: transgenic αKlotho overexposing mouse.

In rats, αKlotho mRNA started to fall 6 hours post-IRI-AKI, and returned to baseline around 3-4 days(37). αKlotho protein in the kidney fell at 3 hours while neutrophil gelatinase-associated lipocalin (NGAL) did not increase until 5 hours, so kidney αKlotho protein is an early marker of AKI(38) (Table 1). Urine and plasma αKlotho paralleled changes in renal αKlotho(38), decreasing dramatically at 3 hours, started to recover ~48 hours and reaching normal levels by 7 days. Soluble αKlotho may be a surrogate for kidney αKlotho.

The first human AKI study showed significantly lower urine αKlotho at the time of AKI diagnosis when compared to healthy controls(38). A larger prospective ICU study showed that AKI patients had lower urinary αKlotho within 48 hours of AKI diagnosis when compared to ICU controls(39). Urine αKlotho concentration and urine αKlotho-to-Cr ratio inversely correlated with hospital and mechanical ventilation days. Each one-fold higher urine αKlotho/Cr was associated with an 83% lower risk of major adverse kidney evets at 90 days(39).

Prognosis.

Heterozygous αKlotho-deficient (kl/+) mice subjected to IRI, CP, sepsis, and UUO had lower αKlotho protein and more kidney damage than wild type (WT) mice(34, 35, 37, 38) (Table 1). Longer ischemia time caused more severe αKlotho deficiency and renal fibrosis. After recovery of kidney function, lower αKlotho associated with more chronic fibrosis and kidney dysfunction(40), suggesting αKlotho level can predict risk of AKI-to-CKD transition (Table 1).

Prophylaxis:

Mice with genetically high αKlotho before AKI had milder kidney damage and better renal outcome in IRI(38) and CP-induced AKI(31). Adenoviral αKlotho gene delivery increased circulating but not kidney αKlotho prior to IRI, ameliorated injury, and kidney dysfunction(37). αKlotho-bearing minicircle vector increased plasma αKlotho, prevented kidney damage in IRI-induced AKI, and attenuated renal fibrosis in an UUO model(41). αKlotho overexpression through CRISPR-Cas9 prevented kidney damage, and alleviated renal fibrosis in CP nephrotoxicity(42) and in UUO(43) (Table 2).

Table 2:

αKlotho protein and gene therapy in animal models of AKI

Animal model	αKlotho manipulation	Blood αKlotho & assay	Urine αKlotho & assay	Renal αKlotho & assay	Comment	Citation
LPS injection (10 mg/kg), mice	αKlotho (0.01-0.02 mg/kg, R&D) IP 60 minutes before LPS injection, once	ND	ND	ND	αKlotho reduced kidney damage in the dose-dependent manner	(48)
IRI (60 min), Wistar rat	Adenovirus encoding mouse αKlotho gene (ad-kl) (1.6 × 1010) 3 days before IRI	ND	ND	ND	1. Ad-KI ↑ αKlotho in the liver, but not in the kidney prior to IRI. 2. Ad-KI ↓ kidney damage and apoptosis	(37)
IRI (30 min), mice	Recombinant αKlotho (lab made) (0.01 mg/kg BW) was IP given after IRI, once	Less ↓ with IB-IP	Less ↓ with IB	Less ↓ with IB	1. αKlotho given immediately after IRI led to better renal recovery 2. αKlotho given at 60 minutes after IRI did not have better recovery	(40)
IRI (30 min), mice	αKlotho protein (lab made) (0.01 mg/kg) for 4 consecutive days starting 24 hours after Bi-IRI.			Less ↓ with IB	αKlotho ↑ renal αKlotho at 20 weeks after IRI, 2. αKlotho did not change CICr at 3 and 7 days, but ↑ it at 8 and 20 weeks. 3. αKlotho ↓ renal fibrosis	(40)
Uni-IRI (30 min) + contralateral Npx for only 24 hours, mice	Minicircle carrying 20 pg αKlotho gene was given using the hydrodynamic tail-vein injection two days prior to AKI induction	Less ↓ with ELISA (Cusabio)	ND	More with IHC	1. Minicircle carrying 20 αg αKlotho gene ↑ plasma αKlotho compared to αKlotho plasmid 2. Minicircle carrying 20 μg αKlotho gene ↓ (1) necrosis, (2) apoptosis	(41)
CP (10 mg/Kg) + 2.0% Pi diet starting 2 weeks after CP injection for 18 weeks, mice	At 4 weeks after cisplatin injection, recombinant αKlotho protein (0.3 mg/kg body weight/month) was given for 16 weeks via osmotic minipumps	ND	ND	Less ↓ with IB and qPCR	αKlotho mitigated cisplatin-induced AKI-to-CKD progression, improved mineral homeostasis, and ameliorated cardiac remodeling and vascular calcification.	(35)
CP (15 mg/Kg) IP once, mice	AAV-dgαKlotho gene was IV injected to Cas9 mice 8 days prior to cisplatin injection	Less ↓ with ELISA (Cusabio)	ND	ND	A CRISPR/Cas9 system sufficiently delivered αKlotho ↑ serum αKlotho, prevented kidney from cisplatin nephrotoxicity, and ↑ survival rate	(42)
UUO up to 7 days, mice	Soluble αKlotho (0.01~0.02 mg/kg BW) was IP given immediately after UUO q.o.d. up to 7 days				αKlotho treatment did not affect the severity of hydronephrosis, but ↓ renal fibrosis probably through binding to TGFpR2	(49)
UUO up to 14 days, mice	αKlotho protein (R&D systems) (0.02 mg/kg BW) was given immediately after UUO q.o.d up to 14 days	ND	ND	ND	αKlotho ↓ renal fibrosis, and ↓ TGF-β1/Smad signaling activity	(44)
UUO up to 10 days, mice	Upon completion of UUO surgery, retrograde ureteral infusion of αKlotho-sgRNA with Cas9 system was performed from proximal site to deliver enzymes for the demethylation of αKlotho gene	ND	ND	Less ↓ mRNA with qPCR	1. Hydroxy-methylation of αKlotho gene ↑ renal αKlotho mRNA 2. Hydroxy-methylation of αKlotho gene ↓ (1) renal fibrosis, and (2) fibroblast and collagen I in the kidney of UUO mice	(43)
UUO up to 10 days, mice	Minicircle carrying 20 μg αKlotho gene was given using the hydrodynamic tail-vein injection two days prior to UUO	Less ↓ with ELISA (Cusabio)	ND	ND	1. Minicircle carrying 20 μg αKlotho gene ↑ plasma αKlotho 2. Minicircle carrying 20 μg αKlotho gene ↓ (1) renal fibrosis, (2) apoptosis, and (3) fibrotic markers in the kidney	(41)
Rhabdomyolysis, IM 50% glycerol (8 mg/Kg), mice	On day 1 post injury, mice received a tail vein injection of 2.0 x 108 EVs containing αKlotho protein in 100 μL or 1 μg/mouse	ND	ND	↑ protein with IB	1. EV containing αKlotho ↓ kidney damage and ↑ kidney recovery 2. ~ 200 folds lower αKlotho than direct IP injection of recombinant αKlotho is required for AKI animal	(45)

AAV: adeno-associated virus; Ad: adenovirus; AKI: acute kidney injury; CKD: chronic kidney disease; CICr: creatinine clearance; CP: cisplatin; dgαKlotho: dead single guide αKlotho; EMT: epithelial mesenchymal transition; EV: extracellular vesicle; IB: immunoblot; IHC: immunohistochemistry; IP: intraperitoneal; IRI: ischemia-reperfusion injury; IP-IB: immunoprecipitation followed by immunoblot; LPS lipopolysaccharide; ND: not done; Pi: phosphate; qPCR: quantitative PCR; Scr: serum creatinine; sgαKlotho: single guide αKlotho; UUO: unilateral ureteral obstruction

Post-AKI treatment:

Clinical utility mandates efficacy if given after AKI. Recombinant αKlotho protein given to mice immediately after IRI, reduced kidney damage(38) but the benefit diminishes dramatically starting one hour after the insult. αKlotho administration immediately after UUO followed up to 14 days reduced renal fibrosis, but not hydronephrosis severity(44). αKlotho-carrying extracellular vesicles(45) promoted kidney recovery in rhabdomyolysis-induced AKI.

At 24 hours post-IRI, when kidney injury was fully established, serum creatinine had peaked, and endogenous αKlotho was at its lowest levels, αKlotho protein injection for four consecutive days preserved endogenous renal αKlotho levels, accelerated recovery, suppressed kidney fibrosis, and protected against AKI-to-CKD transition(40) (Table 2).

Late treatment:

In two CKD models, αKlotho protein was given 4 weeks after CKD induction and sustained for 12 weeks(46), or up to 16 weeks(35). Plasma αKlotho protein levels, kidney function and fibrosis, and cardiac remodeling/cardiomyopathy were improved possibly via both direct effects and secondary effects due to renoprotection (Table 2).

IV. SUMMARY

The database of FGF23 is currently composed largely of human studies, which are strong but only associative in nature. In contrast, the αKlotho database is populated by compelling animal experiments but the clinical data are still in early stages of development. Regarding this pair of proteins with mineral-regulating properties and a plethora of other actions, one can envision many potential applications in human AKI (Figure 1 and Table 3). The potential applications are immense and further research should be directed at the pathobiology of FGF23 in AKI in preclinical studies to dissect whether it is pathogenic or a biomarker (or both); i.e. interventional experiments. Prospective longitudinal clinical studies using simultaneous iFGF23 and cFGF23 assays will enrich our database. Standardization of soluble αKlotho assays will enrich the human database and make it generalizable. The extensive preclinical data in αKlotho are in dire need of human translation in both diagnostics and therapeutics but one faces various hurdles in this effort.

Figure. Protracted natural history of AKI and translational opportunities.

The various stages of AKI are shown on top corresponding to different levels of kidney function. Dx1: Identification of susceptible patients with impending kidney injury. Dx2: Early diagnosis of AKI prior to onset of detectable clinical parameters. Px1: Prognostic predictor of recovery phase-onset and speed of recovery. Px2: Predictor of probability, rapidity, and severity of AKI-to-CKD progression. PPx1: Prevention of onset of AKI in susceptible patients. PPx2: Prevent, delay, or slow down AKI-to-CKD progression. Application in combination with Px2. Rx1: Promote or accelerate recovery from AKI. Rx2: Independent of its effect on kidney function, treatment can be directed to the amelioration of extra-renal complications with cardiovascular complications being a major target.

Table 3:

Potential modalities for FGF23-αKlotho therapy in AKI

Decrease FGF23 bioactivity			Increase αKlotho bioactivity
Mechanism	Description		Mechanism	Description
Decrease synthesis	Phosphate restriction		Increase endogenous protein	Phosphate restriction ACE inhibition PPARγ activator Recombinant αKlotho itself
Increase degradation	GALNT3 inhibitor		Integrated transgene	Successful in animals
Neutralization	Monoclonal antibody		Gene delivery	Successful in animals
Blockade of receptor binding	Experimental development		Recombinant exogenous protein	Successful in animals
Blockade of receptor signaling	Experimental development