Side effects:

Grundlagenforschung zur CNI-Nephrotoxizität

S. Bachmann, Anatomie

DFG FOR 667

DFG FOR 1368

DFG SFB 1365

3 signals to rejection

TCR: Allopeptide on MHC of an APC reacts with TCR of a receptor TC

IL-2: Costimulating molecules of the APC bind to their TC counterpart

(CD80/86 to CD28); activation of CALCINEURIN PHOSPHATASE

->CBM-complex/NFkB, NFAT -> IL2

TC proliferation: IL2/IL2R binding -> mTOR, TC proliferation, clonal proliferation

-> TC mediated rejection

-> TC stimulate BC -> antibody-mediated rejection

B. Rudolph, 2018

C4d

Ca++ and calmodulin -> activation of Cn

Ca2+/Calmodulin-dependent S/T phosphatase

Heterodimer with catalytic subunit isoforms

CnB chain + CnAα, CnAβ, CnAγ

Inhibitors: Cyclosporine A and Tacrolimus/FK506

Immunophilins: (Cyclophilin A,B and FKBP12)

Calcineurin

Immunophilin bound to FK506

(Chaperone; cis/trans isomerase)

Since 1983: CNI

CsA,Tacrolimus,

Immunophilins

Williams & Gooch Exp Rev Mol Med 2012,

Kang CB Neurosignals 2008

CNI and immunophilins

CsA,Tacrolimus,

Immunophilins

Immunophilin

Chaperones

cis/trans Isomerase

Williams & Gooch Exp Rev Mol Med 2012,

Kang CB Neurosignals 2008

Calcineurin has multiple substrates!

CNI – side effects, nephrotoxicity - Pathology

CNI: Short term outcome very good, Risk: early application of high doses-> acute damage

CNI: Long term graft survival suboptimal

1. Functional/ acute arteriolopathy (hyalinosis; 92%)

2. Thrombotic microangiopathy

3. Toxic tubulopathy

4. Interstitial/striped fibrosis

5. Glomerulosclerosis; FGS, FSGS; proteinuria

Mihatsch 2011: CNI sind nephrotoxisch, aber es gibt keine

spezifischen histologischen Läsionen!

Terminus CAN = Chronic Allograft Nephropathy

CNI – side effects, acute nephrotoxicity: Pathology

Transplanted of own kidney:

Afferent constriction

Arteriolar vacuolization of muscular wall

A.A. thrombosis and loop collapse

Isometric tubular vacuolization (ER)

Thrombotic microangiopathy (rare)

Naesens 2018, Leal R et al., KI 2018

M. Thomson, IVA; Figs. a-c GSP

CNI – side effects, chronic nephrotoxicity: Pathology

Late-onset graft dysfunction (grades I to IV):

Arteriolar („beaded“) hyalinisation, subendothelial and media

Leal R et al., KI 2018

Vanhove T and Kuypers D, Transplantation 2017

Pathology – fibrosis and point of no return

Venk, Kriz, Bidani JASN 2015

Pathology: on the origin of fibrosis and its spreading upon AKI

Towards function: adverse effects of CNIs

Cyclosporine and tacrolimus may cause... • Hypertension

• Hyperkalemia

• Hypercalciuria

• Mild metabolic acidosis

• Calcium wasting

• ECM accumulation

• TGF-ß overproduction

• increased apoptosis

• elevated serum creatinine levels

CsA i.v. directly after transplantation: Switch fom diuresis to oliguria

"Between arteriolar constriction and renal scarring"

Morozumi & Mihatsch Transplant Proc 2004

CNI nephrotoxicity – structural vs. functional

View from 2004

Mortensen et al., FIM 2017

View from 2017

Eigene Arbeiten, Berlin

Treating rats, mice, zebrafish, cultured cells

with tacrolimus, CsA

Blankenstein et al. AJP 2017

J. Hu 2018

Juxtaglomerular apparatus

+CNI JG cells Renin

-CNI COX2 +CNI

A reason for COX-2

Höcherl et al. KI 2004

Daily Na excretion

mVal/d x kg b.w.

200

50

Basal Furosemide

Vehicle

Cyclosporine A

Rofecoxib

Cyclosporine A

+ rofecoxib

CsA likely attenuates the natriuretic action of loop diuretics

by inhibition of renal COX-2 expression

A reason for RAAS and CNI (rats, 35 d 15 mg CsA/kg)

Pichler RH… and Johnson R, JASN 1994

ACEI and ARBs – combined with CNI?

TGFß und endothelin levels decrease, creatinine clearance worsened

Unclear, whether co-treatment with ARBs is able to slow the progression of CNI

nephrotoxicity in a human setting

Long term studies: lisinopril and nifedipin have similar beneficial effects,

but also: nifedipin and not lisinopril improved graft function (Naesens)

Na-K-2Cl cotransporter NKCC2

Na-Cl cotransporter NCC

Renal salt handling, extracellular volume

and blood pressure control

Distal nephron and CNI-induced hypertension

CnA isoforms and cyclophilins distributed differentially

FKBP12

CyPA

K+

ROMK

Mg2+ Ca2+

NH4+

Blood

Na+

K+

Na,K-

ATPase

Calcineurin

Urine

P

P

P

Na+,K+,Cl-

Cl-

WNK

Ca2+

Ca2+

Ca2+

Ca2+

SPAK/

OSR1 P P

Cation-coupled chloride cotransporters: NKCC2

K+

ROMK

Mg2+ Ca2+

NH4+

Na+

K+

Na,K-

ATPase

Calcineurin

P

P

P

Cl-

WNK

Ca2+

SPAK/

OSR1 P P CnI Iph.

Na+,K+,Cl-

Blood

Urine

Cation-coupled chloride cotransporters: NKCC2

ΔIs

c,

μA

/cm

²

25µm

*

1

0

-200

-400

-600

-800

-1000

-1200

vehicle cyclosporine A

NKCC2

pT96/pT101-NKCC2

GAPDH

0

100

200

300

*

NKCC2 pNKCC2

CyA

vehicle

*

CyA + furosemide

vehicle + furosemide

GAPDH

pN

KC

C2

sig

na

l, %

isolated perfused TAL

Borschewski A K et al. J Am Soc Nephrol. 2015

Effects of cyclosporine A in rats: NKCC2 activity

0

2

4

6

8

0

20

40

60

80

100

120

140

Na e

xcre

tion

, m

M/k

g b

.w.

BP, m

m H

g

Salt retention Increased Blood Pressure

*

*

CyA

vehicle

10d CyA in rats

Effects of cyclosporine A in rats: volume aspects

CsA stimulates Na,K,2Cl-cotransport

Wu & Vandewalle KI 2000

86Rb+ influx,

nmol.min-1

.mg protein-1

Early findings

in subcultured

mouse mTAL

cells

Conclusion: TAL activated NaCl transport -

a component in CsA – induced hypertension

Bachmann S., Kidney Int 2018

Calcineurin and late distal tubule

Hypertension is salt-sensitive

Tac causes K retention

Hoorn E et al.

Nat Med 7:1304-9, 2011

Effects of tacrolimus in mice: animal physiology

Tac causes Hypertension

Tac increases pNCC Tac increases thiazide-

sensitivity

NCC ablation abrogates

Tac effects

Hoorn E et al.

Nat Med 7:1304-9, 2011

Tacrolimus activates NCC to cause hypertension

Conclusion: DCT activated NaCl transport -

a component in Tacrolimus – associated hypertension

Cn Cn

Calcineurin Inhibitors (cyclosporine A or tacrolimus)

Renal salt retention -> hypertension, electrolyte disorders

Controlling CNI hypertension with diuretics?

Since Schlöndorff et al. JASN 2008: 256 pubmed hits on UPR in the kidney!

SFB C04: Do CNI affect proteostasis (ER stress)?

7 Sites in the UPR for potential intervention

Design: Junda Hu, 2018

ER stress

UPR

Cytoprotective:

↑protein folding

↓protein translation

↑autophagy

Calcineurin Inhibitors

Maladaptive:

↑proapoptotic factors

Toxic tubulopathy 24% in CNI-NT: to study role of UPR

Lhotak AJP F 2012

Lab members and collaborators

Bachmann Lab

Good reviews in the field

are rare, this one is excellent, but needs update!

Naesens Nat Rev Nephrol 2016

CNI & immunosuppressant choice

Basic view in 2006: calcineurin function

Gooch j J, AJP F, 2006

IL-2 to -4 and TNFa

NATURE 1992

IL-2 - The original paper

Venk, Kriz, Bidani JASN 2015

Pathology: on the origin of fibrosis and its spreading upon AKI

CyA; Dr. Rudolph