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Role of Klotho in aging, Phosphate,and CKD

연세 원주의대 병리학교실 엄 민 섭. Role of Klotho in aging, Phosphate,and CKD. John GB, Kuro -o M et al. Am J Kidney Dis. 2011;58:127-34. Kuro -o M. Korean J Intern Med. 2011,26:113-22. Hu MC et al. J Am Soc Nephrol . 2011;22:124-36. Background.

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Role of Klotho in aging, Phosphate,and CKD

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  1. 연세 원주의대 병리학교실 엄 민 섭 Role of Klotho in aging, Phosphate,and CKD John GB, Kuro-o M et al. Am J Kidney Dis. 2011;58:127-34. Kuro-o M. Korean J Intern Med. 2011,26:113-22. Hu MC et al. J Am Soc Nephrol. 2011;22:124-36.

  2. Background • Chronic kidney disease (CKD): state of progressive decrease of kidney function over months or years • Prevalence of CKD: about 13% (USA) • Most CKD Pts. • Die prematurely not from kidney failure, but from early onset of common age-related disease (cardiovascular disease, cancer, and infection …) • Also experience many aging-like symptoms, including hypogonadism, skin atrophy, osteopenia, and cognition impairment

  3. Background • Hyperphosphatemia (high serum P): • Important mortality risk factors in CKD Pts. • Usually evident only in advanced stage of CKD (irreversible state) • ↑FGF-23, ↑PTH, ↓ 1,25 (OH)2Vit.D3 precede hyperphosphatemia during CKD progression Indicate dysregulation of P metabolism ensues in early stage of CKD (long before increase in blood P levels)

  4. Pathogenesis Age-associated decrease in kidney function • One characteristic hallmark of aging: ↓ in physiologic functions of various tissue & organ systems • Kidney • Highly susceptible to aging, and dramatic structural & functional damage than other organ systems • Arterial intimal fibrosis, interstitial fibrosis, glomerulosclerosis, & tubular atrophy

  5. Pathogenesis Age-associated decrease in kidney function • Kidney • Renal fibrosis: • begins as tubulointerstitial injury (↓GFR, ↓renal blood flow) • final common pathologic state of kidney aging and CKD • Aged kidney fail to reverse such fibrotic changes & ultimately develop fibrosis & atrophy due to ↓cellular regeneration & ↑apoptosis (although young kidneys have regenerative capabilities)

  6. Pathogenesis Age-associated decrease in kidney function • Kidney • Dramatic decrease in renal mass (Esp. cortex): > 80 yrs (compared with < 39 yrs) • Renal cortex: significant ↑glomerulosclerosis (> 70 yrs) • Most important biomarker for renal function: GFR • Low (at birth), reach 140 mL/min/1,73m2 (20 yrs, adult level) • A decrease after age 40 yrs, 8 mL (every decade) • <60 mL: state of CKD with high risk renal failure, cardiovascular disease, & death

  7. Pathogenesis Klotho, Phosphate, and Aging • Klotho, in 1997 by Makoto Kuro-o et al. (Nature. 1997:390;45-51) • Named after goddess of Greek mythology who spun the thread of life • As a gene that is mutated in a mouse strain that showed aging-like phenotypes & shortened life span (hypogonadism, growth retardation, accelerated thymic involution, skin atrophy, sarcopenia, vascular calcification, osteopenia, aging lung, cognition impairment, hearing loss, & motor neuron degeneration…)

  8. Pathogenesis Klotho, Phosphate, and Aging • In contrast, transgenic mouse with Klothooverexpression: 20-30% increase in life span • Klotho gene (KL): aging-suppressor gene • KL: encodes single pass transmembrane protein & expressed predominantly in renal tubules

  9. Pathogenesis Klotho, Phosphate, and Aging • InAD hypophosphatemic rickets, FGF-23 gene mutation is identified (resistance to proteolytic degeneration by protease) • Consequently,↑blood FGF-23 (from bone act on kidney to promote phosphaturia) levels • Mice lacking FGF-23: impaired renal P excretion and hyperphosphatemia • FGF-23: Phosphaturic hormone

  10. Pathogenesis Klotho, Phosphate, and Aging • Unexpectedly, FGF-23-deficient micedevelop hyperphosphatemia& aging-like phenotypes resembling KL-deficient mice • FGF-23-deficient = KL-deficient (phenotypically) • Klotho forms a constitutive binary complex with FGF-Rs & function as obligatory co-Rs for FGF-23 (responsible for P homeostasis)

  11. 1,25 (OH)2Vit.D3

  12. Pathogenesis Klotho, Phosphate, and Aging • Importantly, aging-like phenotypes in KL- or FGF-23- deficient mice can be rescued by reversing hyperphosphatemia(low P or low Vit.D diet, knockout of genes for Vit.D action or renal P reabsorption) • Dietary P overload of KL-deficient mouse: rescued by blocking renal P reabsorption • Phosphate retention: responsible foraging-like phenotypes (potential link between aging and P)

  13. Pathogenesis • Endocrine regulation of P metabolism: Vit.D & PTH, well- known regulator of P & Ca DBP: Vit D-binding protein

  14. + FGF23 Endocrine regulation of phosphate homeostasis. Ten years ago, 2 principal calcium-regulating hormones, 1,25 dihydroxyvitamin D3 (vitamin D) and parathyroid hormone (PTH), were thought to regulate phosphate metabolism. PTH increases vitamin D synthesis in kidney (①). Vitamin D in turn decreases PTH (②), thereby closing a negative feedback loop. Now the fibroblast growth factor 23 (FGF-23)–Klotho system has emerged as the principal phosphate-regulating endocrine axes. FGF-23 is secreted from bone and acts on kidney to decrease vitamin D synthesis (③). Because vitamin D increases FGF-23 expression in bone (④), a negative feedback loop exists between FGF-23 and vitamin D. FGF-23 also acts on parathyroid to decrease PTH (⑤). Because PTH increases FGF-23 expression (⑥), another negative feedback loop exists between PTH and FGF-23. Adapted from Kuro-o M. Phosphate and Klotho. Kidney Int. 2011;79(Suppl 121):S20–S23.

  15. Pathogenesis Endocrine regulation of P metabolism • FGF-23(phosphaturic hormone) • Induce phosphaturia by ↓expression of NaPi-2a in renal proximal tubules (PT) • Suppress Vit.Dsysthesisby ↓expression of 1α-hydroxylase (converting to 1,25(OH)2Vit.D3): ↓serum 1,25(OH)2Vit.D3 &↓intestinal P absorption • Negative feedback loop between FGF-23 & 1,25(OH)2Vit.D3 *NaPi-2a: type 2a sodium-dependent phosphate co-transporter

  16. Pathogenesis Endocrine regulation of P metabolism • Klotho • Expressed in distal convoluted tubule (DCT): suggesting primary target of FGF-23 (cf.: FGF-23: ↓NaPi-2a & 1α-hydroxylase, expressed in PT) • 2 possible explanation of this discrepancy (DCT vs. PT) • Low level Klotho is detected in PT, FGF-23 may directly act on PT to suppress NaPi-2a & 1α-hydroxylase • FGF-23 could act on DCT to make them secrete aparacrine factors that act on adjacent PT and suppress NaPi-2a & 1α-hydroxylase (more possible): FGF-23 injection activated FGF pathway only in DCT (Farrow EG et al. JASN 2009;20:955-60)

  17. Pathogenesis Endocrine regulation of P metabolism • In Parathyroid gland (recent study) • Parathyroid gland express Klotho endogenously and act as another major target of FGF-23 • FGF-23 • ↓PTH expression &↓PTH secretion • function as counter-regulatory hormone for PTH • PTH: ? promote FGF-23 expression and/or secretion • Another negative feedback loop between FGF-23 & PTH

  18. Pathogenesis Phosphate metabolism in CKD • High serum Phosphate level • Universal characteristic of patients with CKD • Established independent mortality risk factor • High P: higher risk of death from various diseases, including coronary artery disease • Deranged P metabolism in CKD: dysregulation of FGF-23 & Klotho endocrine axis Kuro-o M. Nephrol Dial Transplant 2009;24:1705-8.

  19. Pathogenesis Phosphate metabolism in CKD • With loss of functioning nephron duringCKD progression, nephron is required to excrete increasing P to maintain serum P level in reference range • ↑FGF-23 levels → Early sign of change in P metabolism in CKD progression • ↓Vit.D(FGF-23: counter-regulatory hormone of Vit.D) → stimulate PTH → 2ndary hyperparathyroidism • Early stage of CKD: ↓ 1,25(OH)2Vit.D3 &↑PTH

  20. Pathogenesis Phosphate metabolism in CKD • Fact “↓Klothoexpression in parathyroid in CKD” • May explain why high FGF-23 fail to suppress PTH in CKD Pts. (FGF-23: suppress PTH in normal human) • ↑FGF-23 & ↓Vit.D→ further ↓Klotho in kidney and parathyroid in CKD Pts. (FGF-23 & Vit.D: can down- & upregulateKlotho) • ↓Klotho →FGF-23 resistance in kidney & parathyroid → ↑FGF-23, ↓Vit.D, and ↑PTH (Vicious cycles) • Pathophysiology of deranged P metabolism in CKD

  21. Vicious cycles ↑ Changes in the phosphate-regulating endocrine system during progression of chronic kidney disease (CKD) . (A) Vicious cycles leading to high fibroblast growth factor 23 (FGF23), high parathyroid hormone (PTH), low vitamin D, and low Klotho in CKD. (B) Increases in serum FGF23 and serum PTH levels and decreases in serum vitamin D and urine Klotho levels precede hyperphosphatemia during CKD progression from stage 1 to stage 5.

  22. Pathogenesis Phosphate metabolism in CKD • In advanced stage of CKD • Increased FGF-23 levels cannot compensate for dietary P overload: overt hyperphosphatemia, Vit.D deficiency, parathyroid hyperplasia • Extremely high level of FGF-23 (biologically active): Activate FGF signaling in tissue → Do not express Klotho and may contribute to systemic complication of ESRD

  23. Recent Advances Secreted Klotho • Klotho on cell surface can be substrate for membrane-anchored protease (α- and β-secretase): release extracellular domain of Klotho into extracellular spaces • Extracellular domain of Klotho can be detected in blood, urine, and CSF • 2 form: Membrane Klotho & secreted Klotho (SK) • Membrane Klotho: obligate co-Rs for FGF-23

  24. 1,25 (OH)2Vit.D3

  25. Recent Advances Secreted Klotho • Cannot act as soluble receptors for FGF-23: because Klotho-FGFR complex (not Klotho or FGFR alone) can bind to FGF-23 with high affinity • Belongs to family I glycosidase • Has homology to β-glucosidase of bacteria & plant • Lactase-phlorizinhydrolase in mammals • Hydrolyze terminal β-glucosidic linkage in saccharide • Recombinant Klotho not show β-glucosidase-like enzyme activity (?)

  26. Recent Advances Secreted Klotho & Ion channels/transporters • SK activates transient receptor potential Ca channel family, specifically TRPV5 & TRPV6 • TRPV5: expressed in distal nephron to mediate renal Calcium ion reabsorption • TRPV6: expressed in intestinal epithelia to mediate intestinal Calcium absorption • May contribute to durable Calcium channel activity and Calcium reabsorptionin distal nephron

  27. Recent Advances Secreted Klotho & Ion channels/transporters • SK regulate K channel activity in kidney • Klotho treatment remove terminal sialic acid residues from N-linked glycan of ROMK1 (renal outer medullary K channel) • ROMK1: expressed in distal nephron & mediate K secretion into urine • Removal of terminal sialic acids: entrapping ROMK1 on cell surface and preventing endocytosis • SK: Promote renal K excretion

  28. Recent Advances Secreted Klotho & Ion channels/transporters • SK directly regulate P transport in proximal tubule by deglycosylation of NaPi-2a: Deglycosylation of N-linked glycan→ NaPi-2a more susceptible to protease in PT brush border membrane →↓number and activity of NaPi-2a → promote phosphaturia (independently of FGF-23) • Secreted Klotho: may be Putative paracrine factors mediating FGF-23 action from PT (? FGF-23 may function as a SK-releasing hormine)

  29. Multiple functions of membrane Klotho and secreted Klotho. Membrane Klotho functions as an obligate coreceptor to fibroblast growth factor 23 (FGF-23) in the kidney and parathyroid gland. This activity ensures a negative phosphate balance by (1) induction of phosphaturia and suppression of vitamin D synthesis in kidney and (2) suppression of parathyroid hormone (PTH) expression and secretion in the parathyroid gland. Secreted Klotho exists in luminal fluid of the renal tubules and in urine and functions as an enzyme that modifies N-linked glycans of the transient receptor potential calcium channel family member TRPV5 and ROMK1 (renal outer medullary K+ channel) on the apical membrane of renal distal tubules, which increases calcium reabsorption and potassium excretion. Secreted Klotho also modifies N-linked glycans of type-2a sodium-dependent phosphate cotransporters (NaPi-2a) on the apical brush-border membrane of renal proximal tubules, which decreases phosphate reabsorptionindependently of FGF-23.

  30. Recent Advances Secreted Klotho & CKD • Possible to measure SK protein in blood & urine (practically difficult to measure in kidney in Pts.) • Progressive↓SK protein during CKD progress (Hu MC et al. JASN. 2011;22:124-36) • ↓urinary Klotho: CKD stage 1-2 in Pts (early stage). • Can be one of most sensitive biomarker for early CKD detection • ↓ SK: ↓ Ca reabsorption & ↑P reabsorption (Klotho activate TRPV5 & inhibit NaPi-2a) • Potentially, contribute to pathophysiology of CKD

  31. Recent Advances Secreted Klotho & CKD • Klotho function as renoprotectivefactors (in animal studies) • Transgenic overexpression of Klotho→ produce dramatic improvement of kidney function and morphologic lesions in mouse model • Injection of recombinant SK or viral vector-transduction: effective in improving serum Cr and decrease apoptosis of ischemic ARF in mouse

  32. Recent Advances Secreted Klotho & CKD • Klotho expression • Down-regulated by angiotensin II • Enhanced by peroxisomeproliferator-activated receptor γ (PPAR γ) agonist (rosiglitazone) • Renoprotective effect • Preserve Klotho expression & interrupt vicious cycles • Angiotensin-converting enzyme inhibitors (ACEI) • PPAR γ

  33. Summary Vicious cycle during CKD progression • ↑FGF-23, ↓Vit.D, ↑PTH, ↓Klotho • Low P-diet & P binder: most good Tx. • Because vicious cycle may be activated long before high P, P-restriction is more effective before serum P & FGF-23 increased • P-restriction: ↓FGF-23, ↑Vit.D, ↑Klothoin kidney & parathyroid • Rosiglitazone & ACEI : up-regulate Klotho

  34. Summary • Klotho in kidney & parathyroid : act as obligatory co-receptors for FGF-23 • FGF-23: create negative P balance (as phosphaturic hormone & counter-regulatory hormone for Vit.D) • Dysregulationof this endocrine axis is linked to progression of CKD and complications • FGF-23 & Klotho: as a novel biomarker & therapeutic targets for CKD (no Tx. for CKD)

  35. Summary • Renoprotective functions of Klotho: hope in therapeutics to improve kidney function • Klotho: has potential to • Serve as a biomarker to diagnose early CKD • Predict progression of kidney disease • Serve as surrogate marker in future clinical trials (Because Klotho in urine decrease with onset & progression of CKD)

  36. Klotho gene polymorphism of G395A is associated with kidney stones • Klotho: novel regulator of Ca & P metabolism • 108 Pts. with renal Ca stone & 51 control Pts. • G395A, C1818T, F325V polymorphism by PCR • Klotho polymorphism: • ↑Ca,↑urine Ca & P • GG type of G395A:≒↑2배 • kidney stones 과 연관 BMD: bone mineral density Telci D et al. Am J Nephrol 2011;33:337-343.

  37. Renin-angiotensin system (RAS) or oxidative stress : Suppress Klothoin kidney • angiotensin II: Klotho억제, ACEI: 신장보호 • Klotho발현과 RAS inCsA induced renal injury • CsATx: ↓Klotho mRNA & protein in mouse (dose-dependent) • Losartan(angiotensin II type I (AT-1) R-blocker): reverse & improve histology • AT1-R blocker: may inhibit aging process by decreasing oxidative stress caused by CsA

  38. CsA7.5: 7.5mg/kg LSRT: losartan for 4weeks Fig. 3. Dose-dependent effect of CsA on expression of Klothorenin in mouse kidney Fig. 6. The effect of angiotensin II blockade on Klotho expression in CsA treated mice

  39. Wnt5A-med. signaling: ↑melanoma metastasis • Wnt signaling: Klotho protein에 의해 억제 • In melanoma cells: • Inverse correlation of Wnt5A & Klotho • rKlotho: Wnt5 internalization & ↓signaling ↑ Wnt5A, ↑ sialidase, ↓syndecan • sialidase inhibitor에의해 억제 가능 • Klotho: invasive potential 감소시킴 (calpain억제 & 결과적으로 filamin cleavage 감소) Camilli TC et al. Pigment Cell Melanoma Research. 2010; 24: 175-86

  40. Insulin & IGF-1: 유방암 증식, 생존, 전이 조절 • Klotho: insulin & IGF-1 억제 (mouse) • IHC: NL (high Klotho) vs. Cancer (very low) (High Klotho: smaller tumor size & low Ki-67) • Cell line study • Forced Klotho expression: 세포 증식 감소 • Silencing of Klotho: 세포증식 증가

  41. Klotho: a tumor suppressor & modulator of IGF-1 & FGF pathways in breast cancer • Cell linestudy • Forced expression of Klotho or treatment with Klotho: IGF-1 & insulin pathway (유방암 증식 인자) 활성을 억제 시킴 • Upregulation of transcription factor CCAAT/enhancer-binding protein β (유방암증식 억제인자) • Klothooverexpression: FGF pathway 활성 • Klotho: potential tumor suppressor, inhibitor of IGF & activator of FGF WolfI et al. Oncogene 2008: 27; 7094-7105.

  42. Large cell neuroendocrine carcinoma • 30 pts of LCNEC (1999-2004), IHC for Klotho • Klotho: New biomarker for good outcome in patients with LCNEC (especially without LN metastasis or lymphatic invasion) Without LN metz (n=14) Without lymphatic invasion (n=11) Usuda J et al. Lung cancer 2011;72:355-59.

  43. Limited-disease small cell carcinoma • 30 pts with SCLC (LD), underwent surgery • Klotho: predictor of favorable outcome of SCLC patients Usuda J et al. Lung cancer 2011 (in press)

  44. Conclusion - Klotho • Anti-aging gene (aging suppressor) • Phosphate metabolism • Associated with CKD progression • Novel biomarker of early CKD & progression • Renoprotective effect • Therapeutic target of kidney disease • Good prognostic factor of variable tumors

  45. 감사합니다

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