1) Complement activation downregulates Klotho expression in renal ischemia-reperfusion injury and delayed graft function through NF-kB signaling in tubular epithelial cells. 2) Inhibition of complement with C1 inhibitor preserved Klotho expression in a swine model of renal IRI and suppressed NF-kB activation. 3) Transplant patients with delayed graft function had significantly lower Klotho expression in allograft biopsies and lower serum Klotho levels long-term, indicating Klotho deficiency may contribute to chronic allograft dysfunction.

1. Klotho in Ischemia/Reperfusion Injury and
Delayed Graft Function
Wisit Cheungpasitporn
October 2, 2015

2. Disclosure
• None

3. The Fates - Klotho (Clotho), Lachesis,
Atropos
• In Greek mythology, three
goddesses determine life span of
every one by controlling the
thread of life.
• Clotho, Lachesis, and Atropos
who spins, measures, and cuts
the thread of life, respectively.
Kuro-o M. Biochim Biophys Acta. 2009; 1790(10): 1049–1058.

4. Klotho gene
• Identified in 1997 , by the Japanese group of Kuro-o et
al.
• Located on chromosome 13q12
• Composed of 5 exons
• Expressed in limited tissues and cell types. The highest
expression is observed in distal convoluted tubules in
the kidney and choroid plexus in the brain
• Lower expression is detected in pituitary gland,
parathyroid gland, pancreas, ovary, testis, placenta,
skeletal muscle, urinary bladder, colon , inner ear , and
breast epithelial cells
Kuro-o M. Biochim Biophys Acta. 2009; 1790(10): 1049–1058.

5. Klotho- deficient mice
7-week-old normal mouse (left) and a klotho mouse, an animal model
that shows multiple phenotypes resembling human aging
Kuro-o M et al. Nature 1997; 390: 45–51

6. Wang Y, Sun Z. Ageing Res Rev. 2009;8(1):43-51.

7. Hu MC et al. Adv Exp Med Biol. 2012;728:126-57.
chromosome 13q12
ADAM 10/17
Glycosidase activity

8. John GB et al. Am J Kidney Dis. 2011;58(1):127-134
Functions of Klotho
Membrane Klotho Soluble Klotho

9. Hu MC et al. Contrib Nephrol. 2013;180:47-63.
Klotho Deficiency in CKD

10. Torres PU et al. Kidney Int. 2007;71(8):730-7.

11. Hu MC et al. Contrib Nephrol. 2013;180:47-63.

12. Clin J Am Soc Nephrol. 2015 Mar 6;10(3):443-51.
EH= essential hypertension, HV= healthy volunteer, RV,
renal vein; RVH, renovascular hypertension
Atherosclerotic renal artery stenosis (n=12) vs Age-matched participants with EH (n=12) vs Healthy volunteers (n=12)

13. Klotho in Acute Ischemia-Reperfusion Injury
(IRI)

14. Hu MC and Moe OW. Nat Rev Nephrol. 2012;8(7):423-9.
IRI- Beneficial Effects of Klotho

15. Aiello S and Noris M. Kidney Int. 2010;78(12):1208-10.

16. Klotho in AKI
Hu MC and Moe OW. Nat Rev Nephrol. 2012;8(7):423-9.

17. Klotho mRNA expression in the kidney after IRI
Sugiura H et al. Nephrol Dial Transplant. 2005;20(12):2636-45.

18. Am J Transplant. 2015 Aug 17. doi: 10.1111/ajt.13415. [Epub ahead of print]

19. Objective
• To investigate the possible involvement of
complement system in the modulation of Klotho
in IRI and in patients affected by DGF.

20. Complement inhibition preserved tubular
Klotho in IRI

21. Wagner E, Frank MM. Nat Rev Drug Discov. 2010;9(1):43-56.

22. Animal model
10 Four-month-old
female large white pigs
Clamping left renal artery
for 30 min
Remove clamping
Control pig
(n=5)
Treated pig with C1 inhibitor
(n=5)
C1-INH diluted in
saline solution
Injection at ear vein
5 min before the end of
ischemia time
Equal volume
of vehicle
Kidney Biopsy
Other names:
Yorkshire pig
Kidney Biopsy

23. Modulation of Klotho in a swine model of renal IRI by
immunohistochemical analysis
Before ischemia
Remarkable reduction of Klotho at
tubular level
Klotho protein was expressed in
renal tubules but not at vascular and
glomerular level.
After 24 h of reperfusion

24. Modulation of Klotho in a swine model of renal IRI by
immunohistochemical analysis
Inhibition of complement by C1-INH
Inhibition of complement by C1-INH
could preserve Klotho expression
Negative control

25. Modulation of Klotho in a swine model of renal IRI by
immunohistochemical analysis
Quantification of specific tubulo-interstitial staining
Down-regulation of Klotho in IRI compared to basal levels and C1-INH group
Western blotting

26. C5a down-regulated Klotho mRNA expression
and
protein production in renal TEC

27. Peng Q et al. J Am Soc Nephrol. 2012;23(9):1474-85.

28. Immortalized Proximal TEC; HK-2 cells
C5a induced a marked reduction of
Klotho mRNA synthesis after 18 hr
and 24 hr of stimulation
A significant decrease in protein
synthesis of Klotho compared to basal
condition at different time points.
qRT-PCR Western blot

29. Complement activation down-regulated Klotho
expression in a NF-kB-dependent manner

30. Izquierdo MC et al. Nephrol Dial Transplant. 2012;27 Suppl 4:iv6-10.

31. Sanz AB et al. J Am Soc Nephrol. 2010;21(8):1254-62.
RelA (p65)
NIK = NF-kB inducing kinase
Pathways for NF-kB activation

32. A striking activation of NIK at
tubular and glomerular level
was detected after 30 min
from reperfusion.
Complement inhibition
by C1-INH led to a
suppression of NIK
Phosphorylation.
Quantification of Total pNIK

33. Low level
before ischemia
Increase of p65/RelA
signal in the cytoplasm
p65/RelA transmigrated into
the nucleus of tubular cells
Immunofluorescence staining for
p65/RelA subunit

34. Immunofluorescence staining for
p65/RelA subunit
C1-INH treated pigs showed impaired p65/RelA activation
both at 60 min and 24 h from IRI

35. Exposure of TEC to C5a in the presence of
CAPE, specific NF-kB inhibitor, in vitro.
NF-kB inhibition prevented C5a-induced Klotho reduction in
TEC as indicated by cytofluorimetric analysis

36. Modulation of Klotho
in
Transplant recipients with DGF

37. Transplant Patients
7 adult first cadaveric
donor renal transplant
recipients with DGF
7 patients with renal biopsy
within 15 days from
transplantation for CNI
A wedge biopsy of donor kidney
performed before transplantation
Immunosuppression
Steroid: 500 methyprednisolone intra-op  prednisone 250mg daily tapered to 25mg by day 8
Anti-CD25 Antibody IV on day 0 and day 4
Mycofenolate 100 mg twice daily
+ cyclosporine A ( C2 levels 800-1200 ng/ml) or Tacrolimus (trough level 8-12 ng/mL)
Graft biopsy performed 7
-15 days after transplant
DGF = the need for dialysis in the first week after transplant
control

38. Modulation of Klotho in human allograft biopsies
Significantly expressed
at tubular level with a
clear distribution along
the basal membrane
Partial reduction in
Klotho expression
Dramatic reduction in
Klotho

39. Modulation of Klotho in human allograft biopsies
• Down-regulation of tissue Klotho expression was statistically significant in
DGF compared to pre-implantation biopsies from the same donors.

40. Klotho serum level
• Transplant patients who experienced DGF had significant low levels of
soluble Klotho compared with patients with EGF after 2 years from
transplantation.
• 8 early graft function
[EGF] patients vs 8 DGF
• eGFR
73.03±14.13 vs. 63.29±20.62;
p>0.05
16 renal transplant recipients 2 years before with
comparable and stable renal function
(8 DGF and 8 early graft function)
Serum Klotho evaluation

41. Discussion
• Complement system is primarily involved in the down-
regulation of Klotho in renal IRI.
• Inhibition of complement prevented the activation of NF-
kB signaling in tubular epithelial cells resulting in
preservation of Klotho production.

42. Discussion
• Transplant patients with DGF presented a significant
down-regulation of tubular Klotho expression resulting
in a permanent deficiency of soluble Klotho after
transplantation.
• Considering the central role of Klotho in preventing
cellular senescence, Klotho deficiency in DGF patients
might play a central role in DGF associated chronic
allograft dysfunction.

43. Hu MC et al. Nephrol Dial Transplant. 2012;27(7):2650-7.

44. Conclusion
• Complement might be pivotal in the down-
regulation of Klotho in IRI leading to a
permanent deficiency after years from
transplantation.

45. Potential applications of Klotho in Clinical
Nephrology
Hu MC et al. Nephrol Dial Transplant. 2012;27(7):2650-7.

46. Hu MC et al. Adv Exp Med Biol. 2012;728:126-57.

47. Klotho administration 30 m post-ischemia–reperfusion injury (IRI)
in acute kidney injury (AKI) rats
Hu MC et al. Kidney Int. 2010;78(12):1240-51.

48. Frémeaux-Bacchi V, Legendre CM. Kidney Int. 2015 [Epub ahead of print]

49. Frémeaux-Bacchi V, Legendre CM. Kidney Int. 2015 [Epub ahead of print]

50. Questions & Discussion

51. Impact of Renal Transplantation and Nephrectomy on
Urinary Soluble Klotho Protein
Kimura T et al. Transplant Proc. 2015 ;47(6):1697-9.

52. Bleskestad IH et al. Eur J Endocrinol. 2015;172(4):343-50.