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

2. Overview Introduction Klotho in Nephrology Conclusions
Calcium and Phosphate metabolism
Vascular and soft tissue calcification
Klotho and FGF23 in CKD
Klotho in AKI
Conclusions
FGF – Fibroblast growth factor

3. Introduction
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

4. Introduction Klotho – 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

5. Introduction 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

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

7. Introduction
Ageing Research Reviews 2009;8:43–51

8. Introduction Japanese researchers observation One year later,
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

9. Introduction
Ageing Research Reviews 2009;8:43–51

10. Introduction
Ageing Research Reviews 2009;8:43–51

11. Introduction
Ageing Research Reviews 2009;8:43–51

12. Introduction 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

13. 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

14. Why is Klotho of particular interest for nephrology?
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
Nephrol Dial Transplant 2007; 22: 1524–1526

15. Effect of FGF23 plus Klotho on renal tubular re-absorption of
inorganic 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 it
In 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.
Nephrol Dial Transplant 2007; 22: 1524–1526

16. Why is Klotho of particular interest for nephrology?
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
Nephrol Dial Transplant 2007; 22: 1524–1526

17. Why is Klotho of particular interest for nephrology?
These observations were highly suggestive of a close cooperation between Klotho and FGF23 and/or its receptor(s)
Nephrol Dial Transplant 2007; 22: 1524–1526

18. Function of Klotho
Nephrol Dial Transplant 2007; 22: 1524–1526

19. 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

20. Regulation of FGF23 signaling by Klotho
Conversion by Klotho of canonical FGF receptor into FGF23-specific receptor
Nephrol Dial Transplant 2007; 22: 1524–1526

22. Regulation of FGF23 signaling by Klotho
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 ; 74(5): 566–570.

23. Regulation of FGF23 signaling by Klotho
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

24. Regulation of FGF23 signaling by Klotho
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
Nephrol Dial Transplant 2009;24: 1705–1708

25. Klotho in Calcium metabolism
Ageing Research Reviews 2009;8:43–51

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

27. 1,25(OH)2D being a major stimulus, which is feedback
regulated 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
Nephrol Dial Transplant 2006; 21: 1770–1772

28. 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
Nephrol Dial Transplant 2009;24: 1705–1708

29. 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
Nephrol Dial Transplant 2009;24: 1705–1708

30. Phosphate regulation by klotho: Hypotheses.
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 Apr;71(8):730-7.

31. The bone–kidney–parathyroid endocrine axes mediated by FGF23 and Klotho
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

32. 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

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

34. 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

35. Klotho in Phosphate toxicity
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

36. Klotho in Phosphate toxicity
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

37. Klotho in Vascular calcification
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 ; 74(5): 566–570.

38. Klotho in Vascular calcification
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 ; 74(5): 566–570.

39. Klotho in Vascular calcification
Wild-type mice with CKD had very low renal, plasma, and urinary levels of Klotho
In humans, authors observed a graded reduction in urinary Klotho starting at an early stage of CKD and progressing with loss of renal function
J Am Soc Nephrol Jan;22(1):

40. Klotho in Vascular calcification
Despite induction of CKD, transgenic mice that overexpressed Klotho had preserved levels of Klotho, enhanced phosphaturia, better renal function, and much less calcification compared with wild-type mice with CKD
J Am Soc Nephrol Jan;22(1):

41. Klotho in Vascular calcification
Conversely, Klotho-haplo insufficient mice with CKD had undetectable levels of Klotho, worse renal function, and severe calcification
The beneficial effect of Klotho on vascular calcification was a result of more than its effect on renal function and phosphatemia, suggesting a direct effect of Klotho on the vasculature
J Am Soc Nephrol Jan;22(1):

42. Klotho in Vascular calcification
In vitro, Klotho suppressed Na(+)-dependent uptake of phosphate and mineralization induced by high phosphate and preserved differentiation in vascular smooth muscle cells
Klotho deficiency contributes to soft-tissue calcification in CKD
J Am Soc Nephrol Jan;22(1):

43. Klotho in Vascular calcification
Klotho ameliorates vascular calcification by
Enhancing phosphaturia
Preserving glomerular filtration, and
Directly inhibiting phosphate uptake by vascular smooth muscle
J Am Soc Nephrol Jan;22(1):

44. Klotho in Vascular calcification
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 ; 74(5): 566–570.

45. Klotho 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

46. Klotho and FGF23 in CKD
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

47. Klotho and FGF23 in CKD
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

48. Klotho and FGF23 in CKD
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
Nephrol Dial Transplant 2009;24: 1705–1708

49. Klotho and FGF23 in CKD
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
Nephrol Dial Transplant 2009;24: 1705–1708

50. Klotho and FGF23 in CKD
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

51. Klotho and FGF23 in CKD
It remains to be determined whether renal Klotho expression levels reflect functional nephron mass that can respond to FGF23

52. J Nephrol 2010; 23(06):

53. J Nephrol 2010; 23(06):

54. J Nephrol 2010; 23(06):

55. Klotho in CKD Klotho is an early biomarker for CKD
Replacement of Klotho may have therapeutic potential for CKD
J Am Soc Nephrol Jan;22(1):

56. Klotho in AKI
AKI diagnosis is based on an increase in serum creatinine or a decrease in urine output
To be effective, treatment of AKI should be started very early after the insult and well before the rise of serum creatinine
Thus, sensitive biologic markers of renal tubular injury in AKI are strongly needed

57. Klotho in AKI
Authors (Hu et al) found that ischemia-reperfusion injury (IRI) in rodents reduced Klotho in the kidneys, urine, and blood, all of which were restored upon recovery
Reduction in kidney and plasma Klotho levels were earlier than that of neutrophil gelatinase-associated lipocalin (NGAL), a known biomarker of kidney injury
Kidney Int Dec;78(12):

58. Klotho in AKI
Patients with AKI were found to have drastic reductions in urinary Klotho
AKI is a state of acute reversible Klotho deficiency, low Klotho exacerbates kidney injury and its restoration attenuates renal damage and promotes recovery from AKI
Kidney Int Dec;78(12):

59. Klotho in AKI
Thus, endogenous Klotho not only serves as an early biomarker for AKI but also functions as a renoprotective factor with therapeutic potential
Kidney Int Dec;78(12):

60. Conclusions
Klotho is an antiaging substance with pleiotropic actions including regulation of mineral metabolism
The mystery of the synergy between Klotho, FGF23 and its receptor has been progressively unravelled

61. Conclusions
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

62. Conclusions
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

63. Conclusions
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

64. Conclusions Klotho is an early biomarker for CKD and AMI
Replacement of Klotho may have therapeutic potential for CKD and AMI

65. Thank You!