Download Physiological oxygenation status is required for fully differentiated

Hélène Simonnet (Villeurbanne)
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Physiological oxygenation status is required
for fully differentiated renal tubule phenotype
- Blood volume regulation
- Glucose homeostasis
- Saving ions and nutrients
- Endocrine: vit D, EPO
- Xenobiotics scavenging
The kidney and its complex
embryonic origin
(Davies JA et al.,
Int J Dev Biol, 1999, 43: 473)
glucose
GLUT1
GLUT2
glucose
ATP
Peu de
biosynthèses
PEP
PLASMA
H+
Na+
acides gras
pyruvate
acetyl-CoA
lactate
isocitrate
malate
glutamate
NH3
αKG
fumarate
URINE PRIMITIVE
glutamine
citrate
OAA
succinate
NADH
NH4+
ATP
I
Na+
II
III
IV
NH4+
V
ANT
SGLT2
Na+
glucose
lactate
O2
glutamine
glucose
GLUT1
GLUT2
glucose
ATP
G6P
PLASMA
H+
Na+
acides gras
Peu de
biosynthèses
PEP
pyruvate
acetyl-CoA
lactate
isocitrate
malate
glutamate
NH3
αKG
fumarate
URINE PRIMITIVE
glutamine
citrate
OAA
succinate
NADH
NH4+
ATP
I
Na+
II
III
IV
NH4+
V
ANT
SGLT2
Na+
glucose
lactate
O2
glutamine
glucose
GLUT1
GLUT2
glucose
ATP
Peu de
biosynthèses
PEP
PLASMA
H+
Na+
acides gras
pyruvate
acetyl-CoA
lactate
isocitrate
malate
glutamate
NH3
αKG
fumarate
URINE PRIMITIVE
glutamine
citrate
OAA
succinate
NADH
NH4+
ATP
I
Na+
II
III
IV
NH4+
V
ANT
SGLT2
Na+
glucose
lactate
O2
glutamine
glucose
acides gras
GLUT1
GLUT2
glucose
aminoacids
Peu de
biosynthèses
pyruvate
acetyl-CoA
lactate
glutamine
citrate
OAA
isocitrate
malate
glutamate
NH3
αKG
fumarate
URINE PRIMITIVE
H+
ATP
PEP
PLASMA
Na+
succinate
NADH
ATP
I
Na+
II
III
IV
aminoacids
LY
NH4+
NH4+
V
ANT
SGLT2
Na+
glucose
lactate
O2
proteins
glutamine
glucose
acides gras
1/2 O2
25-OH vit D
GLUT2
1,25,diOH vit D
glucose
PLASMA
PEP
pyruvate
acetyl-CoA
lactate
glutamine
citrate
OAA
NADH
isocitrate
malate
NADH
NH3
αKG
fumarate
URINE PRIMITIVE
glutamate
succinate
NADH
NADH
ATP
I
Na+
II
III
IV
V
ANT
SGLT2
Na+
glucose
lactate
O2
glutamine
Question: To what extend does hypoxia alter
kidney tubule phenotype?
Approach: High scale analysis of transcriptome
changes after 4 hours of hypoxia
by Restriction Fragment Differential Display
Oxygénation différentielle de suspensions
de tubules isolés (moyenne de 3 préparations)
hyp sévère
140
120
Hyp modéré
pO2
normoxie
100
80
60
40
20
0
1
0
2h
1
4h
6h
production de gluc
hyp sévère
hyp modéré
normoxie
1,50
1,00
0,50
0,00
-0,50
-1,00
-1,50
2h
4h
6h
production de lact
hyp sévère
0,50
hyp modéré
0,40
normoxie
0,30
0,20
0,10
0,00
-0,10
-0,20
-0,30
2h
4h
6h
AAAAAA
TCGA
AGCT
TCGA
AGCT
TCGA
AGCT
CGA
T
Condition 1
T
AGC
NNN
NNN= AAA
TTTTTT
ARNm
ADNc
64 amorces
discriminantes
Séparation des produits de PCR
On peut les identifier grâce à leur
taille et à 11 nucléotides connus
Condition 2
NNN= AAA
On peut détecter un transcrit dans
plusieurs types de PCR
Condition 3
NNN= AAA
RESULTS:
N M S
22%
1%
N M S
8%
1533
N M S
26%
increased decreasedincreased
N M S
decreasedno
bychange
mild
42%
N M S
Kidney specific transcripts.
Transcript (gene, product name)
Atp6v0a1, ATPase, H+ transporting, lysosomal V0 subunit a isoform 1
Response to hypoxia
Decreased
(proximal kidney tubule)
Pklr, L-type pyruvate kinase (normally present in kidney proximal tubule)
Decreased (conf., sq*)
Cyp27b1, 25-hydroxyvitami n D 1-hydroxylase (proximal tubule, cortical
Decreased (conf.,sq*)
segment)
Foxq1, HNF-3 (HFH-1) (renal outer medulla)
Hpse, heparanase (expressed in kidney in inner medulla, in thick ascending
Increased
Increased (conf., rt**)
limb of HenleÕs loop and in distal convoluted tubules, not in proximal tubules)
Scnn1g, amiloride-sensitive sodium channel (regulated by aldosterone)(distal
Increased
tubule, cortical and medulla segments)
Slc12a5, furosemide-sensitive K-Cl transporter (renal outer medulla)
Slc14a1, Urea transporter UT3 (renal medulla)
Slcl1a5, sodium-dependent neutral aminoacid transporter ASCT (normally
Increased
Increased (conf., sq*)
No change
expressed in kidney)
Slc5a2, sodium-dependent glucose transporter SGLT2
No change (conf., sq*, rt*)
(normally expressed in kidney)
Slc38a2, sodium-dependent aminoacid system A transporter ATA2 (widely
expressed in tissues, including kid ney)
No change
transcripts whose products are relevant to epithelial-mesenchymal
transition from tubule cell to myofibroblast.
Transcript (product name)
Response to hypoxia
Myosin subunits (8 found)
Myh1, myosin 2X, muscle type heavy chain, components of fa st-twitch fibers in
Increased
skeletal muscle
Mlc3, myosin II alkali light chain from fast switch skeletal muscle (common exon
Increased
from MLC1-f and MLC3-f)
Myo1b, myosin I heavy chain Myr I (expressed in kidney proximal tubule, increased
Increased
by cell division or protrusion
Mrlcb, myosin regulatory light chain B (predominant in non-muscle cells)
Decreased
Myh9, non-muscle myosin heavy chain-A (normally expressed in kidney)
Decreased (conf., sq*)
Myh10, non-muscle myosin heavy chain-B
No change (conf., sq*, rt*)
Myo5a, non-muscle myosin 5a
Decreased
Myr8, unconventional myosin heavy chain Myr 8b (expressed in developing b rain)
Decreased
Other transcripts generally found in muscle
Myog, myogenin
Increased by mild (conf.,
rt*)
Tnmd, tenomodulin, myodulin
Increased (conf., sq*)
Energy production and storage, nutrient supply
Transcript (product name)
Response to hypoxia
COX2, mitochondrial cytochrome oxidase, subunit 2, mitochondrial genome
No change
Sdha, succinate dehydrogenase A, mitochondrial complex 2, nuclear genome
No change (conf., sq*)
Atp1a1, sodium/potassium ATPase, subunit alpha 1 (normal isoform in kidney)
No change
Glutaminase, kidney type
No change
ND1, mitochondrial complex 1, s ubunit11, mitochondrial genome
Increased
Aldoa, aldolase A
Increased (conf., rt**)
Vegfc, vascular endothelial growth factor C
Increased (conf., rt**)
Ckb, creatine kinase B
Increased
Gatm, L-arginine:glycine amidinotransferase (biosynthesis of creatine, normally
Increased
present in kidney
Gck, glucokinase, hexokinase 4
Slc16a7, MCT2 monocarboxylate transporter (plasma membrane transport of lactate,
Increased
Increased (conf., sq*)
pyruvate, and ketone bodies, normally present in kidney collecting duct)
Pgm1, phosphoglucomutase
Increased by mild (conf., sq)
RGD:708476, SLC25A11, 2-oxoglutarate carrier
Increased by mild (conf., sq)
Ckm, creatine kinase M, oxidative muscle type
Decreased
Amacr, alpha-methylacyl CoA racemase (beta oxidation of b ranched fatty acids)
Decreased
(normally present in kidney tubules)
CONCLUSIONS:
1- Some transcripts are preferentially expressed under mild hypoxia
question: two levels of O2 sensing?
2- Transcripts specific for proximal tubule were down-regulated
whereas those specific for distal nephron or for the medulla zone
were increased by hypoxia.
3- Under hypoxia, nonmuscle myosin transcripts were decreased
whereas those of skeletal muscle-type and that of myodulin
were increased.
Myogenin expression was favored by mild hypoxia.
Shift toward a myofibroblast phenotype under hypoxia
Elodie de Laplanche
Karine Gouget
Jocelyne Demont
Franck Dragounoff
Catherine Godinot
Hélène Simonnet
CGMC, Villeurbanne
CNRS UMR 5534
Loïc Ponger
BBE, Villeurbanne
Guilhem Cléris
CNRS UMR 5558
Naïra Naouar
Guy Perrière
Dominique Mouchiroud
Thomas Hoffmann Qbiogene/MP Biomedicals
Marisa Marquet
Illkirch (France)
TISSUE COHESION
Transcript (gene, product name)
Cd44, Cell surface glycoprotein CD44 (hyaluronate binding protein)
Response to hypoxia
Decreased
Cdh2, N-cadherin (cell-cell adhesion and polarity) (predominant isoform in proximal kidney tubule, absent in other segments)
Decreased (conf, sq*)
Fn1, fibronectin
Decreased (conf.,sq**)
P4ha3, procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide III (rare isoform, synthesis of
Decreased
collagen)
Serpinh1, HSP47, collagen binding protein 2, expressed in distal tubule
Decreased
Thbs4, thrombospondin-4, extracellular matrix protein
Decreased
Itgb4, integrin beta4 subunit precursor
Decreased
Adamts1 (a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, expressed during kidney development
Increased
and in medullary collecting duct)
Ank3, ankyrin3, 190kDa isoform, (outer medulla)
Increased (conf., sq*)
Cntnap1, contactin associated protein (Caspr, axoglial junctions, inhibits cell-cell interactions)
Increased
Gja6, connexin 33 (gap junction membrane channel protein alpha 6, not in kidney, induced in developing neurons by IL7 and bFGF)
Increased
Hpse, heparanase (expressed in kidney in inner medulla, in thick ascending limb of Henle’s loop and in distal convoluted tubules, not in
Increased (conf., rt**)
proximal tubules)
Itga7, integrin alpha chain (H36 alpha 7, expressed during the differentiation of skeletal muscle)
Increased
Lepre1, leprecan, basal membrane protein involved in proteoglycan synthesis, present in muscle and in fibrillar collagen-containing tissues
Increased (conf., sq*)
Mmp2, gelatinase A
Increased (conf.,sq*)
Mmp9, gelatinase B
Increased (conf.,sq*)
Bartomin, (Barmotin, tight junction component)
No change
Col2a1, procollagen, type II, alpha 1
No change
Des, desmin
No change