aristolochic acid nephropathy: from bed to benchside

Aristolochic acid nephropathy: frombed to benchside

F. Debelle, JL. Vanherweghem, J. Nortier

Erasme university hospital Experimental nephrology unitUniversité Libre de Bruxelles

Revisiting AAN…

Revisiting AAN…

1. The Belgian outbreak of « Chinese-herb nephropathy »

2. A worldwide problem

3. Clinical and experimental toxicityof AA

4. Perspectives of research

July 92June 92April 92Dialysis

Creat. 3.0*Renal biopsy**

Creat. 3.8*Renal Biopsy**

Creat. 3.7*Renal Biopsy**

Jan 92

March 92

Creat. 0.8*Creat. 1.1*1989May 90March 91Nov 91

Case n°3Case n°2Case n°1

Exposure to Chinese herbs * mg/dl** Renal biopsy: interstitial fibrosis

1. « Chinese-herb nephropathy »

Vanherweghem JL et al.

Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimenincluding Chinese herbs

Lancet (1993) 341:387-91

Courtesy Dr Baleriaux

«Stephania Tetrandra»

Han Fang-Ji

Vanhaelen et al. Lancet (1994)


Han Fang-Ji


Aristolochic acid I + II(R=OCH3 / H)


Han Fang-Ji



Aristolochia fangchi


Han Fang-Ji



Aristolochia fangchi

Guang Fang-Ji

Potential toxicity of herbal and plant products

Huxtable RJ. Drug Safety (1990)



1. Correct identification … but unknown orunderestimated toxicity




2. Accidental contamination or deliberately modified composition




2. Accidental contamination or deliberately modified composition

3. Misidentification of the plant or substitution by another more toxic compound

Muniz Martinez et al. Nephrol Dial Transplant (2002)

End-stage CHN: evolution of renal replacement therapies

A nnées de su iv i1 9 9 0 1 9 9 2 1 9 9 4 1 9 9 6 1 9 9 8 2 0 0 0 2 0 0 2 2 0 0 4 2 0 0 6

Pat

ient

s A

AN

en

IRT

0

10

20

30

40

50 D ia lys ésT ra n sp la n té sD é c é d é sN b c u m u la tif d e p a tie n ts

Follow-up (years)

Autoradiogram of specific AA-relatedDNA adducts in renal tissue

Schmeiser HH, Arlt VM. Cancer Res (1996)

Arlt VM. Mutagenesis (2002)

Autoradiogram of specific AA-relatedDNA adducts in renal tissue

Schmeiser HH, Arlt VM. Cancer Res (1996)

Arlt VM. Mutagenesis (2002)

AA biotransformation (cytochrome P450)

Revisiting AAN…

Revisiting AAN…


2. A worldwide problem3. Clinical and experimental

nephrotoxicity of AA


The Chenonceaucastle

The green cabinet, Catherine de Médicis’s study

The Brussels

« Aristolochia Tapestry »

(15th century)

Complementary and Alternative Medical (CAM) Therapies

AcupunctureRelaxation techniquesMassageReflexology…

Folk remediesHerbal medicines

= alternative to or complementary to « Western » conventional medicine

The escalating use of alternative therapies

very popular: 47 % of the adult US population (1997), expenditure over $ 21 billion, < 40% disclosed to physicians,

79% of patients perceived the combination to be superior to either one alone

« natural » plant origin safe !!!

traditional medicines: equal official status with Western medicine (Africa, Asia)

dietary supplements (not regulated by the FDA) easy available, over-the-counter, low cost

Eisenberg DM et al. NEJM 1993; JAMA 1998; Arch Int Med 2001

A pharmacy of traditional herbal medicine (Hangzhou, China)

Courtesy Dr F. Debelle

The wide spectrum of indications…

Gillerot et al. Am J Kidney D is (2001)

• Eczema

• Liver enhancement, hepatitis B

• Arthritis, rheumatism

• Pain relief

•…etc…

Aristolochia manshuriensisAristolochia fangchi

Guang Fang-Ji Guan Mutong

FDA recommendations (2001)

Carcinogenicity to humans recognized by the International Agency for Research on Cancer (2002) and the National Toxicology Program (2008)

…

…

…

Gold LS, Slone TH. N Engl J Med (2003)

1 http://www.cfsan.fda.gov/~dms/ds-bot.html

AAN cases around the world

Debelle et al Kidney Int. (2008)

Aristolochia clematitis

Courtesy of Dr B. Jelakovic

The two faces of Janus ?

Chinese herb nephropathy(1992)

SimilaritiesSimilarities

SimilaritiesSimilarities

Renal interstitial fibrosis andurothelial carcinoma

Renal interstitial fibrosis andurothelial carcinoma

Renal interstitial fibrosis, urothelial carcinoma and AA-

related DNA adducts

Renal interstitial fibrosis, urothelial carcinoma and AA-

related DNA adducts

Aristolochic acidspecific DNA

adducts

Aristolochic acidspecific DNA

adducts

Aristolochic acid nephropathy (1996 - )

Aristolochic acid –Balkan endemic nephropathy

(2007 - )

Balkan endemic nephropathy(1956)

Revisiting AAN…

Revisiting AAN…



3. Clinical and experimental nephrotoxicity of AA


Clinical presentation of AAN

Depierreux et al. Am J Kidney Dis 1994

Cosyns et al. Kidney Int 1994

The proximal tubule = target of AA

Markers of structural and functional injury:-- brush border enzymuria

-- microproteinuria

Nortier et al. Kidney Int (1997)

Lebeau C et al. Kidney Int (2001)

Control

10 µmol AA

20 µmol AA

20 µmol AA+ 24h recovery

Control

10 µmol AA20 µmol AA

20 µmol AA+ 24h recovery

ControlControl

ControlControl

15 µM CdCl15 µM CdCl22

15 µM CdCl15 µM CdCl22

0.1% DMSO0.1% DMSO 20 µM AA20 µM AA

0.1% DMSO0.1% DMSO 20 µM AA20 µM AA

Aristolochic acid impedes endocytosis and induces DNA adducts in proximal tubule cells

AA-associated urothelial malignancies

Cosyns et al. Am J Kidney Dis (1999) Nortier et al. N Engl J Med (2000)

Right ureteral tumorRight ureteral tumor

Ureteronephrectomies / AAN patients

2249Total

N Engl J Med 2000; 342: 1686

1839ULB

Am J Kidney Dis 1999; 33: 1011

410UCL

ReferenceN. urothelialcarcinoma

N. patientsCenter

Prevalence +/- 40% !

Alive with no evidence of diseaseLow grade pTa-1

Sudden deathLow grade pTa-1

Dead in generalizationPT3N1-1

Alive with no evidence of disease-pTis5

Alive with no evidence of diseaseHigh grade pTapTis + pTa1

Alive with no evidence of diseaseLow grade pTapTis2

Dead from hepatocarcinomaLow grade pTapTis1

Alive with no evidence of diseaseHigh grade pTa + pTis

pTa1

Alive with no evidence of diseasepTispTis3

Alive with no evidence of diseaseHigh grade pT1pTis1

Alive with no evidence of diseasepTisT11

Dead in generalizationpTisT21

Dead in generalizationHigh grade pT1T31

OutcomeBladderPelvis and/or Ureter

Number of patients

Experimental AAN

Experimental AAN

Chronic AA toxicity in rabbits: A model of Chinese herbsnephropathy?

Cosyns JP et al. Kidney Int (2001)

Experimental AAN

Chronic AA toxicity in rabbits: A model of Chinese herbsnephropathy?

Cosyns JP et al. Kidney Int (2001)

Aristolochic acids induce chronicrenal failure with interstitial fibrosis in salt-depleted rats

Debelle FD et al. JASN (2002)

Debelle F et al. JASN (2002)


CONTROL


CONTROL

AA 10 mg/kg


CONTROL

AA 10 mg/kg Day 35


CONTROL

AA 10 mg/kg Day 35AA 10 mg/kg Day 35

« The severity of the tubulointerstitial damage determines the functional prognosis »

Schainuck et al. Hum Pathol (1970)



« The severity ofdetermines the f

Schainuck




Schainuck

« Injury and matrix remodellingpredict progression »

Halloran P. Necker Seminars (2009)




Schainuck

« Injury and matrix remodellingpredict progression »

Halloran P. Necker Seminars (2009)

Early events promotingfibrosis ?

AA group (10 mg of AA/ kg of bw, sc)

Control group (AA vehicle, sc)

1 2 3 4 5 7 10 14 18 35

Acute phase Chronic phase

Acclimatization

Days of sacrifice

Experimental design

Pozdzik A et al. Kidney Int (2008) Lebeau et al. Nephrol Dial Transplant (2005)

Tubulointerstitial lesions

AA rats: Day 35

X200

X200

*

Control: Day 35

X40

X40 X40

X200 X200

AA rats: Day 5Control: Day 5

Semiquantitative score of tubulointerstitial injury

Phases: Acute Chronic

*p<0.05, **p<0.01, ***p<0.005 AA vs control group

Acute Chronic

Control: Day 3 AA rat: Day 3

x400

x400

AA rat: Day 35

PTEC proliferation Ki-67 immunostaining

Time (days)1 2 3 4 5 7 10 14 18 35

Ki-6

7+ tu

bula

r nuc

lei /

fiel

d

0

10

20

30

40ControlsAA rats

***

***

***

*

***

***

***

*** ***

Phases: Acute Chronic


Dedifferentiation of PTEC NEP / Vimentin double staining

*

Control: Day 3 AA rat: Day 3 AA rat: Day 10

x400

x400

AA rat: Day 10 AA rat: Day 35

Vimentin / Ki-67

Key events of epithelial to mesenchymal transition (EMT)

Zvaifler . Arthritis Research & Therapy 2006

TGF-β alone is capable of inducing epithelial cells to undergo all four steps

Tubular basement membrane integrity Jones stainingControl: Day 2 AA rat: Day 2

x400

x400

AA rat: Day 5

Time (days)1 2 3 4 5 10 35

Base

men

t mem

bran

e de

nuda

tion

scor

e

0

1

2

3 ControlsAA rats

*******Phases: Acute Chronic


X 200

Myofibroblast phenotype: α-SMA immunostaining


X 400

A. Pozdzik et al. Kidney Int (2008)

X 200

Myofibroblast phenotype: α-SMA immunostaining


X 400

A. Pozdzik et al. Kidney Int (2008)

Activation of resident fibroblasts peritubular fibrosis

Renal tissue expression of TGF-βControl: Day 3 AA rat: Day 3

X400

X400

AA rat: Day 10 AA rat: Day 35

Urinary excretion rate of proinflammatory and profibrosing cytokines

Variables Day 10 Day 35

(ng/mmol Cr) Controls AA rats Controls AA rats

IL-1α 8.35 (4.65-9.26)

47.9 b

(23.7-126)8.31

(5.76-17.0)45.7 b

(41.3-87.2)

TNF-α 0.19 (0.00-0.38)

0.00 (0.00-12.4)

0.00(0.00-0.05)

0.28 a

(0.00-1.05)

IFN-γ 0.08 (0.08-1.01)

0.23(0.00-2.19)

0.00 (0.00-0.28)

0.96 a

(0.07-1.54)

MCP-1 119(84.2-145)

219 b

(148-482)74.7

(36.7-110)286 b

(230-490)

IL-4 3.56(0.00-6.00)

9.3(0.00-16.2)

3.31(0.00-3.52)

13.1 b

(3.80-20.7)

Active TGF-β 0.81(0.00-1.91)

56.1 b

(3.20-156)2.20

(1.52-8.41)31.1 b

(9.77-135)

a P < 0.05, b P < 0.005. Pozdzik et al. Nephrol Dial Transplant 2008

Distribution of the monocyte/macrophage infiltrate Semiquantitative score

Distribution of the monocyte/macrophage infiltrate Semiquantitative scoreSemiquantitative score


Phases: Aiguë Chronique Distribution of the monocyte/macrophage infiltrate


Phases: Aiguë Chronique Distribution of the monocyte/macrophage infiltrateDi


Phases: Aiguë Chronique Distribution of the monocyte/macrophage infiltrateDistribution of the mono



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Outer stripe of outer medulla (OSOM)



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Inner stripe of outer medulla (ISOM)



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*p<0.05, **p<0.01, AA vs controls. N = 6 rats/group



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Interstitial T lymphocytesAnti-CD3 Anti-CD20

Control AA Control AA

X400

Anti-CD45RC Anti-CD8Control AA Control AA

Revisiting AAN…

Revisiting AAN…



3. Clinical and experimental nephrotoxicity of AA


Translational nephrology is ongoing !


Physiopathological mechanisms of renal fibrosis, including the potential role of immunocompetentcells



• Therapeutic strategies to reduce the onset

and/or progression of fibrosis



• Therapeutic strategies to reduce the onset

and/or progression of fibrosis

From bench to bedside…

Thank you to…

Nephrology,Pathology andUrology depts, Erasme university hospital, ULBExperimental nephrology unit, ULBInstitute of Pharmacy, ULBInserm U785, Paul BrousseGerman Cancer Institute, Heidelberg, GermanyMolecular Toxicology Unit, Sutton, UKNephrology dept, AZ-VUB

aristolochic acid nephropathy: from bed to benchside

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