P. Dumas, D. Jahns, Ph. Lerch, L. Quaroni, Ch. Quitmann
SR-FTIR : A new hope for patients waiting for
kidney transplantation
D. Bazina, P. Dumasb, Ch. Sandtb, M. Daudonc
a Laboratoire
de Physique des Solides, Université Paris Sud, Bât 510, 91405 Orsay, France
b Synchrotron SOLEIL, Saint-Aubin - BP 48, 91192 Gif-sur-Yvette, France.
c AP-HP, Hôpital Necker-Enfants Malades, Laboratoire de Biochimie A, 75015 Paris, France
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1
Lecture
I/ Generalities on pathological calcifications
II/ Lithiasis
III/ Chemical diversity of intratissular renal calcifications
IV/ When intratissular calcification leads to ESRF
A school case : DHAD
V/ Conclusion & Perspectives
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(B2)
From the medical point of view, pathological
calcifications refer to either a concretion, e.g. a
kidney stone [A], or an ectopic calcification [B]
often associated with tissue alteration. Additionally,
normal physiological calcifications such as bone
may become pathological through the influence of
diseases such as arthrosis or osteoporosis [C].
(C)
(B3)
(A)
(B1)
(A) M. Daudon, P. Jungers, D. Bazin, New England J of Medicine 359 (2008) 101.
(A) M. Daudon, H. Bouzidi, D. Bazin, Urol Res 38 (2010) 459 - 467.
(B1) D. Bazin et al., J. Syn. Rad. 15 (2008) 506 - 509 ; J. Syn. Rad. 17 (2010) 374 - 379.
(B2) P. Dorfmuller, D. Bazin et al., Cardiology Research and Practice (2010) doi:10.4061/2010/685926
(B3) Ch. Nguyen et al., Arthritis & Rheumatism 62 (2010) 2829 - 2830.
(B3) H.K. Ea et al., Arthritis & Rheumatism 63 (2011) 10 - 18.
(C) D. Bazin et al., Osteoporosis Int. 20 (2009) 1065 - 1075.
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Chemical diversity of kidney stones
§ 10% of the population in industrialized countries
Cost of lithiasis in France
900 M€
2l/Day = 300M€
1 cm
Épidémiologie actuelle de la lithiase rénale en France
M. Daudon, Annales d’urologie 39 (2005) 209 - 231.
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Relationship
the macroscopic morphology
&
the chemical composition
of the kidney stone (s)
Pathology (ies)
This relationship can be
- simple i.e.
# one kidney stone made of one chemical phase which is due to one pathology
# a chemical phase in the kidney stone which indicates the pathology
- quite difficult i.e. patient generates several KS containing various chemical
phases, evolution with time of the pathology
M. Daudon, CA Bader, P. Jungers, Urinary calculi – review of classification methods
And correlations with etiology. Scann. Microsc. 7 (1997) 1081 - 1106.
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A Simple Case
Presence of struvite in KS
Pathology :
Infection
Stones containing struvite are considered to be related to urinary
tract infection (UTI).
Struvite kidney stones can grow rapidly over a period of weeks and
can lead to obstruction, hydronephrosis, recurrent pyelonephritis and
decreased kidney function.
In that case, we have an intratissullar infection and thus it is
necessary to give antibiotic for a long time (several weeks).
D. Bazin et al., submitted
Relations between bacterial prints and structural characteristics of struvite-containing
kidney stones at the mesoscopic and atomic scale
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1419cm-1/1035cm-1
apatite with high
carbonatation
rate
Pathology :
Infection
A
2mm
X. Carpentier et al., Urol. 73 (2009) 968 - 975.
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A more complicated case
Several pathologies
CaOx
Calcium oxalate kidney stones may be the result
of at least two pathologies :
- Alimentation disorders (most frequent)
&
- Primary hyperoxaluria type 1
Primary hyperoxaluria type 1 is a rare and very
severe inherited disease leading to recurrent
nephrolithiasis, nephrocalcinosis, systemic oxalosis,
and renal failure, ultimately requiring combined
kidney and liver transplantation.
Peculiar Morphology of Stones in Primary Hyperoxaluria
M. Daudon, P. Jungers, D. Bazin,
New England J of Medicine 359 (2008) 101
Opportunities offered by Scanning Electron Microscopy, Powder Neutron Diffraction and S.R. micro-X-ray
Fluorescence in the study of whewellite kidney stones,
M. Daudon, D. Bazin, P. Jungers, G. André, A. Cousson, P. Chevallier, E. Véron, G. Matzen,
J. App. Cryst. 42 (2009) 109-115.
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The case of a concretion (Kidney
stone) on a ectopic calcification
(Randall’s plaque)
[B]
[A]
Randall’s plaques
M. Daudon, O. Traxer,
J.C. Williams, D. Bazin
Book’s chapter, Springer-Verlag,
in press.
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The case of a concretion on a ectopic
calcification
Stone morphology suggestive of RP
M. Daudon, O. Traxer, P. Jungers, D. Bazin
Renal Stone Disease 900 (2007) 26 - 34.
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PACC
Sursaturation of Ca and (PO4)
X. Carpentier et al., J. Syn. Rad. 17 (2010) 374 – 379.
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Lecture
I/ Generalities on pathological calcifications
II/ Lithiasis
III/ Chemical diversity of intratissular renal calcifications
IV/ When intratissular calcification leads to ESRF
A school case : DHAD
V/ Conclusion & Perspectives
Workshop on IR spectroscopy,
P. Dumas - Synchrotron Soleil, (F), D. Jahns, Ph. Lerch, L. Quaroni, Ch. Quitmann, Paul Scherrer Institute
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Calcifications present in/on tissues
SR-FTIR
Signal to noise ratio & spatial resolution (5x5 mm).
Sensitivity (very small samples)
Revisiting the chemical diversity of intratissular calcifications
P. Dumas et al., Synchrotron infrared microscopy at the French Synchrotron Facility SOLEIL, Infrared
Physics & Technology (2006) 49 : 152 - 160.
L.M. Miller, P. Dumas, From structure to cellular mechanism with infrared
Microspectroscopy, Curr Opin Struct Biol. 2010 Aug 23.
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Chemical diversity of KS
Chemical diversity of ITC
Table 1 gathers the different chemical
phases identified in this study.
Samples
B85
BR165
Épidémiologie actuelle de la lithiase rénale en France
M. Daudon, Annales d’urologie 39 (2005) 209 - 231.
BR166
BR167
BR175
BR177
BR178
BR179
BR180
BR181
BR 182
BR184
BR189
BR191
BR193
Chemical phase identified
Acid methyl1 Uric
Sodium hydrogen urate monohydrate,
CA, Whitlockite
DHA
C1
OCP
Amorphous Silica,
DHA
DHA
DHA
DHA
CA, OCP
C1
C1
Calcite, C1
CA
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Octacalcium phosphate
1119 cm
-1
BR182-4_a
4000 3600 3200 2800 2400 2000 1600 1200 800
-1
cm
Apatite1033 cm-1
OCP 1119 cm-1 & 1037 cm-1
OCP - Ca8H2(PO4)6 is a thermodynamically
metastable phase and it transforms to apatite
spontaneously.
From a physiological point of view, this
compound has been proposed to participate as a
precursor of biological apatites. Also, regarding
kidney stones, this compound has been found in
pregnant women for which the metabolism of Ca
is modified in order to build the bone network of
the foetus.
For these thermodynamic and physiological
reasons, localization of OCP in kidney tissue
indicates that the lithiasis process is an active one
suggesting a significant increase of the Ca
metabolism.
M. Iijima, et al. (1998) Effects of Ca addition on the formation of octacalcium phosphate and apatite in solution at pH7.4 and
at 37°C, J. of Crystal Growth 193 : 182 – 188.
M. Iijima et al. (1992) J. Crystal Growth 116 : 319.
O. Suzuki et al. (2008) Conversion of OCP into Hydroxypatite and bone regeneration, Key Engineering Materials 361-363 :
993 - 996.
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Sodium Hydrogen Urate Monohydrate
1,2
1
3600 cm-1
Transmitance
0,8
0,6
1004 cm-1
0,4
0,2
BR165COUPE1B_a
4000 3600 3200 2800 2400 2000 1600 1200 800
-1
cm
0
The presence of sodium urate in tissues is not really a surprise. This chemical compound
has been found in the chemical composition of RP.
Anyway, there is an interesting feature regarding this chemical compound and its
interaction with calcium oxalate. Different studies have underlined the fact that increasing the
concentration of urate promotes the crystallization of calcium oxalate in human
urine. Let’s just recall that nowadays, calcium oxalate (CaOx) is the main component of more
than 70% of all stones in Western countries. The presence of urate in kidney tissue constitutes
thus a major issue.
P.K. Grover et al. (1992) Calcium oxalate crystallization in urine and glycoaminoglycans, Kidney international 41 ; 149 – 154.
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Silica
Silica has an exogen origin.
-1
Transmitance
1102 cm
BR177 Cong Pos 5_a
4000 3600 3200 2800 2400 2000 1600 1200 800
-1
cm
K.M.Kim et al. (1983) Siliceous deposits in human urinary calculi – an
E.M.Study, Urological research 11 ; 155 - 158.
M. Augusti et al. (1993) Calcul urinaire de silice secondaire à l’absorption de
gélopectose chez l’enfant. Progres en urologie 3 ; 812 - 815.
F. Wang et al. (2009) Oxidative stress contributes to silica nanoparticucle –
indueced cytotoxicity in human embryonic kidney cells, Toxicology in vitro
23 ; 808 – 815.
M. Zhu et al. (2011) A mesoporous silica nanoparticulate/β-TCP/BG composite
drug delivery system for osteoarticular tuberculosis therapy, Biomaterials 32 ;
1986 – 1995.
As noticed by K.M. Kim et al., if
silica calculi in man are extremely rare,
siliceous deposits in urinary stones
appear to be more common than
generally recognized.
Silica may come from excipient
associated to drugs or be the result of
metabolism of drug itself. Magnesium
trisilicate administration is prescribed
in the case of stomach disorders. Of
note, the presence of silica has been
underlined in children after adsorption
of gelopectose.
Finally, several papers have been
investigated the silica toxicity, this
material being develop as drug
delivery system.
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Whitlockite
Whitlockite in KS is an infrequent
component and has been associated with
chronic urinary tract infection in 100% of
calculi from women.
-1
1080 cm
-1
1025 cm
Shoulders
-1
980 & 1155 cm
BR165COUPE1C_a
4000 3600 3200 2800 2400 2000 1600 1200-1 800
cm
R. Lagier et al. have reported that
whitlockite deposits have been also noticed
in infection such as tuberculosis as well as
non infection conditions, such as in aortic
media, cartilage and bone tissue. Is the
presence of whitlockite in tissues constitutes
a signature of infection too?
Another important relationship
As reported by A.S. Parker et al., the analysis
of data suggest a positive association of UTI
history with renal cell carcinoma
development.
L. Maurice-Estepa et al. (1999) Crystalline phase differentiation in urinary calcium phosphate and magnesium phosphate calculi, Scan
J. Uril Nephro. 33 : 299 – 305.
R. Lagier et al. (2003) Magnesium whitlockite, a calcium phosphate crystal of special
interest in pathology, Pathology, Research and Practice 199 : 329 – 335.
A.S. Parker et al. (2004) History of urinary tract infection and risk of renal cell carcinoma, Am. J. of Epidemiology 159 ; 42-48.
JK McLaughlin et al. (1984) A population-based case control study of renal cell carcinoma J. Natl Cancer Inst. 72 ; 275 – 284.
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BR183
Metabolisation of drug : the case of Foscarnet.
Foscarnet
(phosphonoformic
acid),
a
apyrophosphate analogue that inhibits the
DNA polymerase of al herpes viruses is
commonly used in patients with acquired
immunodeficiency syndrome (AIDS).
BR183
– glomérule
et tubes
Glomerule
& tubule
Vibration at 936 cm-1 is specific
of the P-O-Na group and attests
the presence of the unchanged trisodium
foscarnet salt
BR183_10c_glomerule-a
1,2
Transmitance
1
0,8
0,6
0,4
0,2
G. Zanetta et al., Transplantation 67 (1999) 1376 -1378
0
4000 3600 3200 2800 2400 2000 1600 1200 800
-1
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BR183
– glomérule
et tubes
Glomerule
& tubule
When we consider now the proximal tubule,
this fingerprint disappears and absorption
bands are now associated to apatite
BR183_10c_glomerule-a
1,2
1
BR183_14c_Paroitube_Apatite_a
0,8
1
0,6
0,8
0,2
0
4000 3600 3200 2800 2400 2000 1600 1200 800
-1
cm
Transmitance
0,4
-1
1004 cm
0,6
0,4
0,2
0
4000 3600 3200 2800
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2400 2000 1600 1200 800
-1
cm 20
Lecture
I/ Generalities on pathological calcifications
II/ Lithiasis
III/ Chemical diversity of intratissular renal calcifications
IV/ When intratissular calcification leads to ESRF
A school case : DHAD
V/ Conclusion & Perspectives
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When intratissular calcification leads to ESRF (End Stage Renal Failure)
In France, the number of kidney
transplantation is around 2800 per year.
Among these patients, 10 to 15% of them the
diagnostic is not established
At the same time, adenine phosphoribosyltransferase
(APRT) deficiency is an under recognized
(heterozygote between 0,4 et 1,1% of the population),
autosomal recessive disorder of adenine metabolism,
leading to 2,8-dihydroxyadeninuria that causes
nephrolithiasis and kidney failure in a significant
proportion of untreated patients.
http://www.rarekidneystones.org/dha/physicians.html
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Epidemiology of APRT
Countries
Finland, Greece, Hungary, Iraq,
# 170 cas
The Netherlands, Poland, Turkey
Belgium, Switzerland, Tchéquie
Spain, UK
Austria, Canada, Arabia Saudi.
USA
Germany
Islande
France (Bollée, JASN, 2010 + M.D. Necker)
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2
3
4
6
7
27
58
23
Clinic case
In september 2009, a 64 year-old woman was admitted to Necker
hospital for rapidly progressive renal insufficiency failure with a serum
creatinine increased up to 512 µmol/l giving an estimated glomerular
filtration rate of 10 ml/min/1.73 m2.
Her past medical history revealed well controlled hypertension
diagnosed 10 years ago, and chronic renal failure diagnosed 2 years
ago with a serum creatinine of 215 µmol/l, initially attributed to
nephroangiosclerosis.
Because no obvious cause for her rapid
worsening of her renal failure was found,
a kidney biopsy was performed, showing
an extensive tubulo-interstitial fibrosis
scattered with multiples small crystals.
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2,8 DHAD
Some difficulties & traps
Such spherical cristallites
can be easily mixed up with
other chemical compounds
namely
- amorphous urates
- some drugs
- or ammonium urate
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Made the right decision
Such possible mistake has dramatic consequences because the
patient does not received the specific drug (allopurinol) able to
stop crystallization.
In a number of cases, the evolution of the kidney function
tends toward end-stage renal failure (ESRF).
Moreover, we can underline the major risk to destroy any
further grafted kidney.
At the opposite, allopurinol, which is a therapy for life, cannot
be given blindly due to possible severe side effects.
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Of note, irrespective of the human cost, the financial cost of
an undiagnosed case leading to ESRF is at least:
-50,000€ a year for dialysis (the average time before
kidney transplantation is about 18-24 months)
-50,000€ for the kidney graft (around 2800 kidney
transplantations per year)
-5 to 10,000€ per year of medical therapy,
management of side effects and follow-up of the patient.
By comparison, the cost of 8h synchrotron to establish a
clinically relevant diagnosis is 4000€ (Less than one hour is
sufficient)
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2,8 DHAD
FT-IR
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2,8 DHAD
Diagnostic is quite difficult due the small number of crystals as
well as their size quite small.
a
SR- FT-IR
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2,8 DHAD
Biopsy
Au/glass
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2,8 DHAD
PARAFFINE
CRISTAUX 2,8 DHAD
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Conclusion
SR-FTIR constitutes a perfect technique to characterize
pathological calcifications in kidney tissues :
Take home message 1
Several new chemical phases have been identified : silica,
urate, OCP. SR FTIR constitutes definitely a significant
opportunity for nephrologists in order to understand at the
cellular level dysfunctionnement of kidney
Take home message 2
For patients waiting a kidney transplantation, a
diagnostic through SR-FTIR constitutes a significant
opportunity to understand the etiology of their renal failure.
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Aknowledgments
P. Jungers, B. Lacour Necker Hospital
O. Traxer, J.P. Hayman, Tenon Hospital
P. Conort, I. Tostivint Lapitié Salpétrière Hospital
F. Lioté, Dr. K.Ea, Ch. Nguyen, Ch Chappard, Lariboisiere Hospital
M. Mathonnet, Limoges Hospital
X. Carpentier, Nice Hospital
G. Matzen, E. Veron, F. Fayon, M. Allix, CRMHT, Orleans
E. Foy, LPSue, CNRS-CEA
P.A. Albouy, B. Fayard, S. Rouziere, LPS
A. Lebail, Lemans University
G. André (LLB)
S. Reguer, D. Thiaudière, S. Mocuta (Soleil-Synchrotron)
This work was supported by the Physics and Chemistry
Institutes of CNRS and by contract ANR-09-BLAN-0120-02.
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