Molecular Mechanisms of Pediatric Kidney Disease

Nader Gordjani MD PhDProfessor of Pediatrics

Universities of Freiburg and Frankfurt

Molecular origins of renal diseases

H2O

Hypophasphatemic rickets

Bartter-Syndrome

Hypokalemic alkalosis:

Gitelman-S.

Diabetes insipidus renalis

NEPHROTIC SYNDROME ?

CYSTÎC KIDNEYS ?

kidney Glomerular structure

Protein loss

Lesions sites of the glomerulus

Filtration barrier

TH-Lypmphocytes

Capillaries

Mesangial cells

s. Biopsie

Deposits of antibodiesand complement

Podocyte

Autoantibodies

Nephrotic Syndrome

Nephritic Syndrome

kidney

Proteinuria > 40 mg/m2 * h

> 1000 mg/m2 * 24 h

Plasma Albumin < 25 g/l

Nephrotic Syndrome in Children

80% Steroid-sensitive

Stable function

20% Steroid-resistant

deterioration

Nephrotic Syndrome in Children

Somlo S Nat Genet 24: 333 (2000)

The glomerulus in health & NS

Somlo S Nat Genet 24: 333 (2000)

Morphology of the Filtration Barriere

normal

Nephroc syndrome

Geheimnisse des „Podozyten“ Molecular structures of the Podocyte

What is the role of the podocyte in nephrotic syndrome ?

Chemokines

cytokines

complement

autoantibodies

Inflammatoryagents

Das kongenitale NS vom finnischen Typ Nephrin

Congenital nephrotic syndrome Finnish Type

Familiäres Steroid-resistentes NS Podocin

Familial Steroid resistant Nephrotic Syndrome

Die familiäre Glomerulosklerose

a-act 4

Familial Glomerulosclerosis

What is the role of the podocyte in nephrotic syndrome and as target of immunmodulation?

Chemokines

cytokines

complement

autoantibodies

Inflammatoryagents

Imm

unos

uppr

essi

on

Ca2+- Signalling in the cell

Stimulation of:

Signalling pathwaysGene expressionChannel opening/closing …

0.0

1.0

2.0

3.0

fluo

resc

en

ce r

atio

34

0/3

80

ATP 10-4 M

C a2+ 10-6 M

3 m in

Calcium signalling in the Podocyte

• Fluorescence microscopy with Fura-2 AM Single-Photon TubeVideo-ImagingLaser-Scanning

RT-PCR• Isolation of rat glomeruli by sieving method• Statistical analysis: student´s t-test

Mouse Podocytes in Culture

33o C 37o C

CCR

3

CCR

4

CCR

1

CCR

2

CCR

5

GAP

DH

334

605

409

563

410

334

500bp

CC-receptors of cultured mouse podocytes

chemokine receptor EC50 [ng/ml]

Rantes 1, 3, 5 50-700

TECK 4 300

Eotaxin 3 50

MIP-1 1, 5 20

MIP-1 5 100

MCP-1 2 10

MCP-2 2 200

MCP-3 1, 2 200

TARC 4 10

CXC

IL-8 1, 2 200

0.0

1.0

2.0

3.0flu

ores

cenc

e ra

tio 3

40/3

80

ATP 10-4 M

C a2+ 10-6 M

3 m in

0.0

0.5

1.0

1.5

2.0flu

ore

scen

ce r

atio

34

0/3

80

ATP 100 µm ol/l

R AN TES 0.4 µg/m l

M C P-1 0.2 µg/m l

M IP-1 60 ng/m l

5 m in

0.0

0.2

0.4

0.6

0.8

1.0flu

ores

cenc

e ra

tio 3

40 /

380

nm

400 700 1000

RANTES

ng / m l

(n=39) (n=11) (n=4)

0.0

0.1

0.2

0.3

0.4flu

ore

sce

nce

ratio

34

0 / 3

80

nm

M C P 1 M IP 1 Eotaxin IL - 8-

R AN TES 0.7µg/m l

ATP 10-4M0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5flu

ores

cenc

e ra

tio 3

40/3

80

C a2+ 10-6 M

3 m in

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

f

luor

esce

nce

rat

io 3

40/3

80 n

m

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

f

luor

esc

ence

rat

io 3

40/

380

nm

(n=5) (n=8)

CyA

ATP A II

CyA

(n=6)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

flu

ore

sce

nce

ra

tio 3

40

/38

0 n

m

RANTES 700 ng/m l

CyA 10 -5 M

Freshly isolated rat glomerulum

25 µm 25 µm

decapsulated

25 µm 25 µm

decapsulated

Immunofluorescence staining for podosynapsinin freshly isolated rat glomerulum

25 µm

Immunofluorescence staining for WT-1in freshly isolated rat glomerulum

Freshly isolated rat glomerulum

0

0.5

1

1.5

2

2.5

3

3.5

4flu

ores

cenc

e ra

tio 3

45/3

80 n

m

AC H 1 µm ol/l

M C P-1 20 ng/m l

10 m in

C YA 10 nm ol/l

R AN TES 70 ng/m l

ConclusionsChemokines induce an increase of [Ca2+]i in mouse podocytes by release from cytosolic Ca2+-stores and thus stimulate Ca2+-mediated signal transduction.

Therefore podocytes are target cells of these proinflammatory factors.

ATP also causes a characteristic rise of [Ca2+]i, which is mediated by Ca2+-influx from the extracellular space in addition to store release.

Cyclosporine A did not influence the chemokine- or ATP-associated Ca2+-effects.

These findings could partly be confirmed in podocytes from freshly isolated intact rat glomeruli.

Physiology

Andreas BenesicRuth FreudingerMichael GekleGerald Schwerdt

Pediatrics

Nader GordjaniAntje KirchhoffBrigitte Wollny

University of Würzburg

University of Freiburg

Physiologie

Rainer GregerHermann PavenstädtJens LeipzigerRoland NitschkeViktoria Munzinger

Rainer Greger1946 - 2004

Institute of Physiology/Freiburg

What causes Cyst formation in kidneys?

… and damage in other organs?

M. Waters et al. Pediatr Nephrol (2011) 6:1039–1056

… and other organs?

M. Waters et al. Pediatr Nephrol (2011) 6:1039–1056

Hildebrandt F et al. JASN 2009;20:23-35

Juvenile/infantile Nephronophthisis

Hildebrandt F et al. JASN 2009;20:23-35

Ciliar defects: a unifying theory of cystic kidney disease?

Ciliar defects: a unifying theory of cystic kidney disease?

Lin et al. PNAS 2002

Subcellular localization of nephrocystins

Hildebrandt F et al. JASN 2009;20:23-35

Hildebrandt F et al. JASN 2009;20:23-35

Chapin et al. JCB 2010

Ciliar defects: a unifying theory of cystic kidney disease?

Hildebrandt F et al. JASN 2009;20:23-35

Correct mitotic spindle

orientation

False mitotic spindle

orientation

Hildebrandt F et al. JASN 2009;20:23-35

The hedgehog signaling pathway may be involved in renal cystogenesis.

Hildebrandt F et al. JASN 2009;20:23-35