Klotho in CKD and AKI Dr CKD – Chronic Kidney Disease AKI – Acute Kidney Injury

Klotho in CKD and AKIKlotho in CKD and AKI

DrDr

CKD – Chronic Kidney DiseaseAKI – Acute Kidney Injury

OverviewOverview

► IntroductionIntroduction►Klotho in NephrologyKlotho in Nephrology

Calcium and Phosphate metabolismCalcium and Phosphate metabolism Vascular and soft tissue calcificationVascular and soft tissue calcification Klotho and FGF23 in CKDKlotho and FGF23 in CKD Klotho in AKIKlotho in AKI

►ConclusionsConclusions

FGF – Fibroblast growth factor

IntroductionIntroduction

►Klotho One of the three goddesses of

the Moirae*, who in Greek mythology ►Control the life (spins the thread

of life) and destiny of everyone

She is the goddess who helps life to unfold, in contrast to ►The (apoptotic) goddess Atropos,

who cuts the thread of life

*Moirae - any of the three Greek goddesses of fate or destiny


►Klotho – medical scienceKlotho – medical science►Gene

Identified app. 13 years ago, by the Japanese group of Kuro-o et al.*

Named after Greek goddess,►As prolonging lifespan is probably the most

important role of this ageing-suppressor gene

*Nature 1997; 390: 45–51


►Japanese researchers reported Defect in Klotho gene expression in the

mouse resulted in a syndrome that resembled ►Human ageing, including

Short lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis and emphysema

►The gene encoded a membrane protein that Shared sequence similarity with the beta-

glucosidase enzymes

Nature 1997; 390: 45–51


7-week-old normal mouse (left) and a klotho mouse, an animal model that shows multiple phenotypes resembling human aging


Ageing Research Reviews 2009;8:43–51


►Japanese researchers observation Led them to conclude that

►The protein might function as part of a signalling pathway involved in the regulation of ageing and related diseases

►One year later, The same group isolated the human

homologue of the Klotho gene and determined its structure

Nature 1997; 390: 45–51 Biochem Biophys Res Commun 1998; 242: 626–630








►The Klotho gene encodes Single-pass transmembrane

protein ►Belongs to a family 1

glycosidase ►Expressed primarily in renal

tubules (distal tubules)

Present in the circulation and urine

Blood Cells Mol Dis 1998; 24: 83–100

Why is Klotho of particular interest for nephrology?

►Klotho is involved in the Renal control of calcium, phosphate and

vitamin D metabolism Suppresses phosphate re-absorption in

renal proximal tubule, by directly binding to FGF receptors

Regulates Ca2+ re-absorption in the distal convoluted tubule by ►Stabilizing the TRPV5 Ca2+ channel in the

plasma membrane

Nephrol Dial Transplant 2007; 22: 1524–1526


►KlothoKlotho Inhibits renal 1-alpha 25 hydroxylase

activity and thereby ►Decreases circulating calcitriol levels

Therefore appears to ►Synergize with the renal tubular effects of

parathyroid hormone (PTH) on Ca2+ and phosphate transport, whereas

►Antagonizes the stimulatory effect of PTH on calcitriol synthesis by the kidney


Effect of FGF23 plus Klotho on renal tubular re-absorption ofinorganic phosphate (iP) and synthesis of 1,25 dihydroxy vitamin D(calcitriol).

FGF23 + Klotho act synergistically with PTH to reduce tubular iP re absorption. However, FGF23 + Klotho inhibit tubular calcitriol synthesis, in contrast to PTH which stimulates itIn end-stage renal disease (ESRD), the physiological inhibition of tubular iP re-absorption by both PTH and FGF23 + Klotho becomes ineffective. The concomitant increase in PTH secretion leads to excessive iP release from bone into the extracellular space. The clinical consequence is hyperphosphataemia.



►Klotho-deficient mice and FGF23-deficient mice have an identical phenotype including Hyperphosphataemia, hypercalcaemia,

elevated plasma calcitriol and vascular calcification, in addition to premature ageing

►In contrast, over-expression of the Klotho gene Extends the lifespan and increases resistance

to oxidative stress



►These observations were highly suggestive of a close cooperation between Klotho and FGF23 and/or its receptor(s)


Function of KlothoFunction of Klotho


Regulation of FGF23 signaling by Klotho

►The common phenotypes of Klotho and FGF23 overexpression and deletion, respectively, led to the postulate of a Common signal transduction pathway

►Kurosu et al.* showed that Klotho protein directly bound to multiple

FGFRs The Klotho–FGFR complex bound to FGF23

with higher affinity than FGFR or Klotho alone

*J Biol Chem 2006; 281: 6120–6123


►Conversion by Klotho of canonical FGF receptor into FGF23-specific receptor



►Klotho functions as an obligatory coreceptor for FGF23 Experimental studies have demonstrated

convincingly that FGF23, in the absence of klotho, cannot exert its bioactivities►For instance, despite extremely high serum

levels of FGF23 (about 2000-fold higher) in klotho ablated mice, FGF is unable to exert its phosphaturic effects in these mice

Kidney Int. 2008 ; 74(5): 566–570.


►The fact that FGF23 requires Klotho as a co-receptor explains Why Klotho-deficient mice develop

phenotypes identical with those observed in FGF23-deficient mice and

Why Klotho-deficient mice had extremely high serum FGF23 levels

Nephrol Dial Transplant 2009;24: 1705–1708


►Kidney-specific expression of Klotho explains why FGF23 can identify the kidney as its target organ among many other tissues that express multiple FGFR isoforms


Klotho in Calcium Klotho in Calcium metabolismmetabolism


Pflugers Arch - Eur J Physiol 2010; 459:333–343

1,25(OH)2D being a major stimulus, which is feedbackregulated by klotho via an inhibition of the 1-hydroxylase activity.

Klotho has a significant impact on the Ca 2+ reabsorption via the epithelial Ca 2+ channel, TRPV5, in the distal convoluted and connecting tubules

Klotho is produced in the kidneys and is reduced in chronic uraemia

Mutations in the klotho gene and klotho deficiency have been related to the process of ageing, osteoporosis, rteriosclerosis, ectopic calcifications and skin atrophy


Klotho in Phosphate metabolism

►The bone–kidney endocrine axis mediated by FGF23 and Klotho has emerged as an essential component in the regulation of phosphate homeostasis When phosphate is in excess, FGF23 is secreted

from bone and acts on the kidney where Klotho is expressed

As a phosphaturic hormone, FGF23 reduces the amount of sodium phosphate co-transporter type-2a (NaPi-2a) on the brush border membrane of proximal tubules, thereby promoting renal phosphate excretion


Klotho in Phosphate metabolism

►As a counter-regulatory hormone for vitamin D, FGF23 suppresses synthesis and promotes inactivation of 1,25- dihydroxyvitamin D3 in proximal tubules


PhosphaPhosphate te

regulatioregulation by n by

klotho: klotho: HypothesesHypotheses

.. The first arrow starts from the intestine, where a reduced dietary phosphate intake diminishes serum phosphate concentration and leads to a decrease in PTH secretion, which physiologically reduces urinary phosphate excretion. In addition, to save phosphate, the renal action of FGF23 will decrease facilitating tubular phosphate reabsorption by the stimulation of sodium-dependent phosphate cotransporters (NPT2a and NPT2c). It will also facilitate the synthesis of 1,25(OH)2D3 in spite of low PTH levels. The increase in calcitriol levels stimulates sodium-dependent phosphate cotransporter type IIb expression and intestinal phosphate absorption. Then, to counteract the activation of these three phosphate-saving mechanisms and to avoid hyperphosphatemia, the renal synthesis of klotho is increased. This increase in renal klotho will facilitate the phosphaturic action of FGF23. Klotho binds to FGFR1(IIIc) and forms the specific FGF23 receptor. Furthermore, klotho negatively regulates the synthesis of 1,25(OH)2D3 by enabling FGF23 binding to its receptor and thereby its inhibitory effect on 1--hydroxylase activity. At the bone level, klotho could stimulate bone resorption and phosphate release by acting on TRPV5, which is a recently identified osteoclast function modulator. The increased levels of 1,25(OH)2D3 could also stimulate osteoclast differentiation and bone resorption and thereby phosphate release. It could also stimulate skeletal FGF23 synthesis to control further, at the renal level, any excessive increase in serum phosphate resulting from the activation of the prophosphatemic mechanisms. Abbreviations: PTH, parathyroid hormone; FGF23, fibroblast growth factor-23; TRPV5, epithelial calcium channel TRPV5 (transient receptor potential vallinoid-5).

Kidney Int. 2007 Apr;71(8):730-7.

The bone–The bone–kidney–kidney–

parathyroid parathyroid endocrine endocrine

axes mediated axes mediated by FGF23 and by FGF23 and

KlothoKlotho

Active vitaminD(1,25-dihydroxyvitaminD3) binds to the vitamin Dreceptor (VDR) in osteocytes. The ligand-boundVDRforms a heterodimerwith a nuclear receptor RXRand transactivates the FGF23 gene expression.FGF23 secreted from bone acts on the Klotho–FGFR complex in kidney (the bone–kidney axis) and parathyroid gland (the bone–parathyroid axis).In kidney, FGF23 suppresses synthesis of active vitamin D by down-regulating expression of the Cyp27b1 gene and promotes its inactivation by up-regulating expression of the Cyp24 gene, thereby closing a negative feedback loop for vitamin D homeostasis. In the parathyroid gland, FGF23 suppresses production and secretion of PTH. Since PTH is a potent inducer of Cyp27b1 gene expression, suppression of PTH by FGF23 reduces expression of the Cyp27b1 gene as well as serum levels of 1,25-dihydroxyvitamin D3, which closes another long negative feedback loop for vitamin D homeostasis. Klotho and FGF23 are indispensable for the regulation of vitamin D metabolism because defects in either Klotho or FGF23 cause hypervitaminosis D. Nephrol Dial Transplant 2009;24: 1705–1708

Overlay of the systemic phosphate homeostasis with the major players in the phosphate regulatory network and their functional interactions(-stimulation; inhibition)

Laboratory Investigation 2009;89, 7–14

Pflugers Arch - Eur J Physiol 2010; 459:333–343

Klotho in Phosphate toxicity

►Disruption of the bone–kidney endocrine axis mediated by Klotho and FGF23 results in hyperphosphataemia, hypercalcaemia and hypervitaminosis D associated with multiple ageing-like phenotypes These observations have raised the

possibility that toxicity of phosphate, calcium and/or vitamin D may be responsible for the premature ageing syndrome observed in Klotho- and FGF23-deficient mice


►Recent epidemiological studies support the notion of ‘phosphate toxicity’ in humans Serum phosphate levels were shown to

positively correlate all-cause mortality risk, even when serum phosphate levels are within the normal range

Circulation 2005; 112: 2627–2633


►In addition, Chronic kidney disease (CKD) patients

with hyperphosphataemia (≥6.5 mg/dl) were reported to have higher risk for death resulting from several diseases including coronary artery disease than those with the lower serum phosphate levels (<6.5 mg/dl)

J Am Soc Nephrol 2001; 12: 2131–2138

Klotho in Vascular Klotho in Vascular calcificationcalcification

►Both FGF23 and klotho ablated mice develop extensive vascular and soft tissue calcification

►Inability to clear the required amount of phosphate by the kidney, due to the absence of FGF23-klotho activity, leads to Increased serum accumulation of phosphate

in these genetically modified mice, causing extensive calcification

Kidney Int. 2008 ; 74(5): 566–570.


►Serum calcium levels are also elevated in both FGF23 and klotho ablated mice

►Moreover, increased sodium phosphate co-transporter activity in both FGF23 and klotho ablated mice increases renal phosphate reabsorption which in turn can facilitate calcification

Kidney Int. 2008 ; 74(5): 566–570.


►Wild-type mice with CKD had very low Wild-type mice with CKD had very low renal, plasma, and urinary levels of renal, plasma, and urinary levels of KlothoKlotho

► In humans, authors observed a graded In humans, authors observed a graded reduction in urinary Klotho starting at reduction in urinary Klotho starting at an early stage of CKD and progressing an early stage of CKD and progressing with loss of renal functionwith loss of renal function

J Am Soc Nephrol. 2011 Jan;22(1):124-36.


►Despite induction of CKD, transgenic Despite induction of CKD, transgenic mice that overexpressed Klotho had mice that overexpressed Klotho had preserved levels of Klotho, enhanced preserved levels of Klotho, enhanced phosphaturia, better renal function, phosphaturia, better renal function, and much less calcification compared and much less calcification compared with wild-type mice with CKD with wild-type mice with CKD



►Conversely, Klotho-haplo insufficient Conversely, Klotho-haplo insufficient mice with CKD had undetectable levels mice with CKD had undetectable levels of Klotho, worse renal function, and of Klotho, worse renal function, and severe calcificationsevere calcification The beneficial effect of Klotho on vascular The beneficial effect of Klotho on vascular

calcification was a result of more than its calcification was a result of more than its effect on renal function and effect on renal function and phosphatemia, suggesting a direct effect phosphatemia, suggesting a direct effect of Klotho on the vasculatureof Klotho on the vasculature



► In vitro, Klotho suppressed Na(+)-In vitro, Klotho suppressed Na(+)-dependent uptake of phosphate and dependent uptake of phosphate and mineralization induced by high mineralization induced by high phosphate and preserved phosphate and preserved differentiation in vascular smooth differentiation in vascular smooth muscle cellsmuscle cells Klotho deficiency contributes to soft-tissue Klotho deficiency contributes to soft-tissue

calcification in CKDcalcification in CKD



►Klotho ameliorates vascular Klotho ameliorates vascular calcification by calcification by Enhancing phosphaturiaEnhancing phosphaturia Preserving glomerular filtration, and Preserving glomerular filtration, and Directly inhibiting phosphate uptake by Directly inhibiting phosphate uptake by

vascular smooth musclevascular smooth muscle



►Collectively, these observations bring new insights into our understanding of the roles of the FGF23 -klotho axis in the development of vascular and soft tissue calcification

Kidney Int. 2008 ; 74(5): 566–570.

Klotho and FGF23 in CKDKlotho and FGF23 in CKD

►The National Kidney Foundation task force indicated that The cardiovascular mortality of a 35-year-

old patient on dialysis was equivalent to that of an 80-year-old healthy individual, rendering CKD to be the most potent accelerator of vascular senescence

J Am Soc Nephrol 1998; 9: S31–S42


►Furthermore, the American Heart Association announced in 2003* that CKD should be included in the highest risk

group for cardiovascular disease►Like Klotho-deficient mice,

CKD patients suffer vascular calcification and have elevated serum levels of FGF23 and phosphate►Importantly, Klotho expression is decreased in

CKD patients**

*Circulation 2003; 108: 2154–2169,**Biochem Biophys Res Commun 2001; 280: 1015–1020


►These observations suggest that Klotho deficiency may contribute to pathophysiology of CKD

►Of note, recent animal studies have shown that Klotho functions as a renoprotective factor Although the mechanism remains to be

determined, over-expression of Klotho ameliorated progressive renal injury in mouse models of glomerulonephritis* and acute kidney injury** Nephrol Dial Transplant 2009;24: 1705–1708

*Proc Natl Acad Sci USA 2007; 104: 2331–2336**Nephrol Dial Transplant 2005; 20: 2636–2645


►Thus, decrease in Klotho expression potentially accelerates renal damage, leading to a deterioration spiral of Klotho expression and renal function Because 1,25- dihydroxyvitamin D3

increases Klotho expression in kidney, vitamin D treatment may be useful for interrupting this vicious cycle



►Epidemiological studies have identified high serum levels of phosphate and FGF23 as independent mortality risks in CKD patients

► Importantly, serum FGF23 levels increase before serum phosphate levels increase during the progression of CKD suggesting that Resistance to FGF23 may be one of the earliest

changes in phosphate metabolism in CKD►Although the mechanism of FGF23 resistance is yet to

be determined, it can be caused by a decrease in renal Klotho expression



►Provided that serum FGF23 levels are a surrogate marker for renal Klotho expression levels, the fact that high serum FGF23 levels are associated with poor prognosis in patients undergoing dialysis Suggests that low renal Klotho expression

levels may be primarily responsible for the poor prognosis


►It remains to be determined whether renal Klotho expression levels reflect functional nephron mass that can respond to FGF23

J Nephrol 2010; 23(06): 619-625

J Nephrol 2010; 23(06): 619-625

J Nephrol 2010; 23(06): 619-625

Klotho in CKDKlotho in CKD

►Klotho is an early biomarker for CKDKlotho is an early biomarker for CKD

►Replacement of Klotho may have Replacement of Klotho may have therapeutic potential for CKD therapeutic potential for CKD


Klotho in AKIKlotho in AKI

►AKI diagnosis is based on an increase AKI diagnosis is based on an increase in serum creatinine or a decrease in in serum creatinine or a decrease in urine outputurine output

►To be effective, treatment of AKI To be effective, treatment of AKI should be started very early after the should be started very early after the insult and well before the rise of serum insult and well before the rise of serum creatininecreatinine Thus, sensitive biologic markers of renal Thus, sensitive biologic markers of renal

tubular injury in AKI are strongly neededtubular injury in AKI are strongly needed


►Authors (Hu Authors (Hu et alet al) found that ischemia-) found that ischemia-reperfusion injury (IRI) in rodents reperfusion injury (IRI) in rodents reduced Klotho in the kidneys, urine, reduced Klotho in the kidneys, urine, and blood, all of which were restored and blood, all of which were restored upon recoveryupon recovery Reduction in kidney and plasma Klotho Reduction in kidney and plasma Klotho

levels were earlier than that of neutrophil levels were earlier than that of neutrophil gelatinase-associated lipocalin (NGAL), a gelatinase-associated lipocalin (NGAL), a known biomarker of kidney injuryknown biomarker of kidney injury

Kidney Int. 2010 Dec;78(12):1240-51.


►Patients with AKI were found to have Patients with AKI were found to have drastic reductions in urinary Klothodrastic reductions in urinary Klotho

►AKI is a state of acute reversible AKI is a state of acute reversible Klotho deficiency, low Klotho Klotho deficiency, low Klotho exacerbates kidney injury and its exacerbates kidney injury and its restoration attenuates renal damage restoration attenuates renal damage and promotes recovery from AKIand promotes recovery from AKI

Kidney Int. 2010 Dec;78(12):1240-51.


►Thus, endogenous Klotho not only Thus, endogenous Klotho not only serves as an early biomarker for AKI serves as an early biomarker for AKI but also functions as a renoprotective but also functions as a renoprotective factor with therapeutic potentialfactor with therapeutic potential

Kidney Int. 2010 Dec;78(12):1240-51.

ConclusionsConclusions

►Klotho is an antiaging substance with Klotho is an antiaging substance with pleiotropic actions including regulation pleiotropic actions including regulation of mineral metabolism of mineral metabolism

►The mystery of the synergy between The mystery of the synergy between Klotho, FGF23 and its receptor has Klotho, FGF23 and its receptor has been progressively unravelledbeen progressively unravelled


►It has become increasingly clear that phosphate metabolism plays a critical role in the pathophysiology in CKD and that

►Hyperphosphataemia should be aggressively treated to improve life expectancy of CKD patients In this context, the bone–kidney–parathyroid

endocrine axis mediated by Klotho and FGF23 is expected to be a novel target of therapeutic intervention


►Hypothetically, enhanced FGF23-klotho activity can delay the process of calcification by negatively impacting the serum phosphate balance Further studies will determine if the

pharmacological manipulation of FGF23 activity can be beneficial in fine tuning existing treatments of vascular and/or soft tissue calcification


► FGF23 - Klotho plays a central role in the pathogenesis of altered mineral metabolism and secondary hyperparathyroidism in CKD patients

► FGF23 - Klotho can be used not only as a biomarker for assessing phosphate retention but also as a predictor of mortality and future development of refractory hyperparathyroidism Further elucidation of FGF23 function and

regulation will help to establish a more rational approach for the management of the mineral and bone disorders that are associated with high burden of morbidity and mortality in CKD patients


►Klotho is an early biomarker for CKD Klotho is an early biomarker for CKD and AMIand AMI

►Replacement of Klotho may have Replacement of Klotho may have therapeutic potential for CKD and AMItherapeutic potential for CKD and AMI

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Klotho in CKD and AKI Dr CKD – Chronic Kidney Disease AKI – Acute Kidney Injury