Application Note #MSI-05

Getting a Better Understanding of Pharmacodynamics& Toxicity in Kidney by Quantitative Mass Spectrometry

Imaging

Would you evaluate the candidate drug efficacy by following companion biomarkers related to

glomerulonephritis and the drug itself in fine histological kidney regions such as glomeruli?

Is there a biomarker associated with kidney small substructures (tubules, ducts…), kidney dysfunction

(inflammation, necrosis…) due to a disease, stress or drug toxicity?

IntroductionIntroduction

The kidney serves several essential regulato-

ry roles in the human body and the dysregulation of

renal physiological properties can induce serious

pathologies. Moreover, it could be a major site of

organ damage caused by drug toxicity (i.e. nephro-

toxicity). The kidney can be separated into three

major parts; the cortex, the medulla and the pelvis

with functional significance. These regions have

several small subregions of a few micrometers scale,

such as for example the renal corpuscles (glomeruli),

the tubules, the loops of Henle or the collecting

ducts. There are different classes of lipids with speci-

fic role in the kidney cell proliferation, the cellular

signaling or the inflammation process. For instance,

dysfunction of sphingolipids (SL) and glycerosphin-

golipids (GSL) metabolism induces the accumula-

tion of these molecules in kidney substructures (e.g.

glomeruli) which could result in different kidney

diseases [1], such as polycystic [2], cancer, diabetic

nephropathy, Fabry disease [3], glomerulosclerosis

or nephritis.

Classical mass spectrometric analysis coupled

with liquid chromatography (LC-MS) uses tissue

homogenates, cannot provide spatial data. However,

Mass Spectrometry Imaging (MSI) permits simul-

taneous detection and quantification of a wide range of

molecules without labelling (drugs, exogenous and

endogenous metabolites, lipids, peptides or proteins)

while keeping their spatial information at the low

micrometer level. So, knowing the distribution and the

quantification of these molecules, such as the specific

lipids in small histological kidney substructures, will

be useful in drug development to improve understan-

ding of pharmacodynamics (physiologic kidney

changes induced by the drug on its local environment)

and of toxicity (drug-induced kidney abnormalities).

In this Application Note, several lipid biomarkers with

key roles in renal diseases and with precise localiza-

tion in the kidney substructures will be illustrated.

al diseases and with precise localiza-

ey substructures will be illustrated.

1

ate drug efficacy by following companion bioma

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Imaging

2

Lipid distribution in kidney transversal sections from wild type rat; zoom on renal cortex region for optic and molecular

images showing co-localization of glomeruli (black arrows) and GM3 related Species. Mass accuracy measurement are

reported for each lipid shown in the table, differences between experiment and theoretical values are expressed in ppm

(part per million).

FigureFigure

Experimental Sectionntal SectionExperiment

Animal: Kidneys from wild type (male wistar strain)

rat were removed; snap frozen and stored at -80°C.

Sectioning: Kidneys were sectioned following trans-

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HM560 cryostat (Thermo Scientific, Germany) at

-20°C and mounted on ITO conductive glass slides

(Delta Technology USA).

Matrix: 9AA (9-Aminoacridine) powder (100 mg)

was used and vaporized on tissue sample using

home-built sublimation apparatus (200°C, 12 min,

2.10-3 mbar).

Mass spectrometry imaging: Solarix 7.0T FTICR

(Bruker Daltonik, Germany) with SmartBeam II

laser. Positive mode (600-1800 Da) at 60 µm spatial

resolution.

Software: FlexImaging 4.0 (Bruker Daltonik,

Germany) & Quantinetix 1.4 (ImaBiotech, France).

m/z 699.497 m/z 747.5182 m/z 766.5392 m/m z// 6666666666666666666666666666666666999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999......................................4444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444499999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999997777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777 m/m z// 7777777777777777777777777777777777777777777777777444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444444777777777777777777777 777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777..................................5555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111188888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888882222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222 m/m z// 7666.......5555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333333399999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999999222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222222

Results & discussion

Gangliosides (GL) are molecules composed

of a glycosphingolipid (ceramide and oligosaccha-

ride) with one or more sialic acids (e.g. n-acetylneura-

minic acid, NANA) linked on the sugar residue. GL

are considered as biomarkers in numerous diseases

targeting especially the brain such as Tay-Sachs,

Gaucher or Sandhoff diseases. In these diseases, GL

levels increase because of alteration or degradation of

enzyme function [7]. One therapy could be to repair

the enzymatic function [8]. NANA-Gal-Glc-ceramide

abbreviated as GM3 (G for ganglioside, M for mono-

sialic acid with only two sugar moieties) is detected in

kidney section using MSI. As presented in the insert

of the figure, GM3 related species are highly co-loca-

lized with glomeruli, which are a network of capilla-

ries that performs the first step of blood filtering.

Some dysfunction of glomeruli properties may induce

renal pathology such as glomerulo- nephritis or

sclerosis [9]. Three forms of GM3 species have been

detected in glomeruli with specific fatty acid chain

lengths (18:0, 20:0 and 22:0). All these chemical

structures have been confirmed by MS/MS measure-

ment and by high mass accuracy measurement (below

ppm level).

The above figure shows the distribution of

eight lipids, related to defined lipid classes, such as

phosphatidic acid (PA), phosphatidylglycerol (PG),

phosphatidylethanolamine (PE), phosphatidylserine

(PS), phosphatidylinositol (PI), sulfatides (ST),

gangliosides (GL) and cardiolipins (CL). The nega-

tive detection mode allows observing specific lipid

species, but the combination of both polarities give

access to other classes, such as phosphatydilcholine

(PC), ceramide (Cer) or sphingomyelin (SM). Each

lipid has a specific distribution in the histological

structures of the kidney, especially in the cortex and

the inner/outer medulla as shown on H&E staining.

For example, PI (38:4) has a homogenous distribu-

tion in the cortex and the outer medulla region. From

this kind of lipid profiles in tissue, it would be

possible to study and characterize their lipid metabo-

lism in animal disease models (e.g. diabetic versus

wild-type model) by following: 1) specific up/down

regulation: 2) the modulation of the fatty acid chain

length or 3) the degree of saturation of the fatty acid

residues.

Sulfatides (ST) have a role in myelin func-

tion and stability [4]. Dysfunction of ST homeostasis

may be involved in kidney disorders [5]. For

instance, ST levels are increased in human renal cell

carcinoma and altered in a mouse model of polycys-

tic kidney disease. It has been demonstrated that ST

are up regulated in cancerous tissue and therefore be

considered as an interesting oncometabolite. Varia-

tions in ST levels could also be associated with

cardiovascular defects induced by renal failure and

with kidney dysfunction due to protein overload

nephropathy. As we can see on the above figure, ST

(32:1) is primarily concentrated in the whole medulla

with a higher intensity in the inner medulla region.

Cardiolipins (CL) appear as an exception in

the classical lipid family due to their structure contai-

ning four fatty acid residues. Unlike other lipids

localized in cellular/subcellular membrane, CL are

an important component of the inner mitochondrial

membrane. CL are considered as a mitochondrial

lipid signature [6] and consequently, as a mitochon-

drial dysfunction related disease biomarker. Ion

corresponding to CL (72:8) is detected in the cortex

region of the kidney but also in glomeruli and tubule

as presented in the above figure.

Conclusions & summaryConclusions & summary

High spatial resolution MSI allows the detec-

tion and quantification of a wide range of lipids in

specific histological kidney substructures unlike

classical LC-MS. Some of the histological related

lipids observed using MSI, such as gangliosides

(glomeruli), cardiolipins (cortex) or sulfatides

(medulla) are considered as reliable biomarkers of

disease state. They permit the evaluation of the

efficiency or the potential toxicity of a treatment. The

detection of theses markers within tissue section, in

combination with the precise distribution of the drug

candidate in these different kidney substructures

provides huge amount of information in support of

PK/PD studies.

ImaBiotech provides crucial information about

the efficiency or potential toxicity of your drug

candidate, thanks to the detection of specific renal

disease biomarkers which are localized in fine

histological kidney substructures.

& discussionResults &

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MS Imaging Department | 885 ave. Eugène Avinée - 59120 Loos - France | +33 (0) 320 164 090 | contact@imabiotech.com

References

1. Sparvero, L.J., et al., Mapping of phospholipids by MALDI imaging (MALDI-MSI): realities and expectations. Chemistry and Physics of

Lipids. 165(5): p. 545-562.

2. Ruh, H., et al., MALDI imaging MS reveals candidate lipid markers of polycystic kidney disease. Journal of Lipid Research, 2013.

3. Roy, S., et al., Imaging mass spectrometry: a new tool for the analysis of skin biopsy. Application in Fabry's disease. Ann Pharm Fr, 2006. 64(5):

p. 328-34.

4. Marsching, C., et al., Imaging of complex sulfatides SM3 and SB1a in mouse kidney using MALDI-TOF/TOF mass spectrometry. Anal

Bioanal Chem, 2011.

5. Stettner, P., et al., Sulfatides are required for renal adaptation to chronic metabolic acidosis. Proceedings of the National Academy of Sciences.

110(24): p. 9998-10003.

6. Wang, H.Y., S.N. Jackson, and A.S. Woods, Direct MALDI-MS analysis of cardiolipin from rat organs sections. J Am Soc Mass Spectrom,

2007. 18(3): p. 567-77.

7. Whitehead, S.N., et al., Imaging Mass Spectrometry Detection of Gangliosides Species in the Mouse Brain following Transient Focal Cerebral

Ischemia and Long-Term Recovery. PLoS One. 6(6): p. e20808.

8. McEachern, K.A., et al., A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease.

Molecular Genetics and Metabolism, 2007. 91(3): p. 259-267.

© 2013 ImaBiotech SAS

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ferencesRefe

Sparvero, L.J., et al., Map1. Sparve

Characterization of kidney disease

Lipid companion biomarkers evaluation at the level of drug localization

Renal disease state markers discovery in specific kidney tissue substructures

High spatial and high spectral resolution imaging

Molecular histology combines with classical histology techniques

KeywordsKeywords

Companion biomarkers

Mass spectrometry imaging

Renal disease

Nephropathy

Gangliosides

Cardiolipins

Sulfatides

Sphingolipids

Glomerulus

AuthorsAuthors

Hamm Gregory

Legouffe Raphael

Viala Jean

Poncet Laetitia

Heron Alain

Stauber Jonathan