Comunicazione intercellulare nel glomerulo renale

1. Comunicazione intercellulare nel
glomerulo renale
Rastaldi Maria Pia, MD, PhD
Fondazione D’Amico per la Ricerca sulle Malattie Renali
& Fondazione IRCCS Policlinico,
Milan - Italy
Torino, 23/01/2009

2. The kidneys together are receiving roughly 20%
of cardiac output.
The entire blood volume passes through the
renal filter every 5 min.
Therefore, glomerular cells are constantly exposed to an enormous variety
of stimuli (blood pressure and content).
In this continuously varying environment, communication between
glomerular cells becomes essential to the maintenance of a correct
function of the glomerular filtration barrier.
Studies on this specific field are difficult because of the extremely
convoluted structure of the renal glomerulus, but we can expect advances
in the near future, given the enormous progresses in technologies that
make it feasible the creation and subsequent analysis of animal models.

3. Podocytes are highly specialized ramified cells that enwrap the
glomerular basement membrane.
Their processes are connected to each other by a specialized
intercellular junction, called slit diaphragm.

5. Synaptic-like communication: a novel signaling hypothesis
In the last years, several studies have shown that podocytes have numerous
similarities with neuronal cells.
Both cells are in fact highly ramified, they have a common cytoskeletal
organization and a common machinery of process formation.
Furthermore, a number of expression-restricted proteins, such as nephrin,
densin, dendrin, glomerular epithelial protein 1 (GLEPP1), the aminoacid
transporters CAT3 and EEAT2, the cytoskeletal proteins synaptopodin and
drebrin, and the RNA processing protein Sam68-like mammalian protein 2, are
shared by the neuron and the podocyte.

6. Our group has contributed to this line of research by showing that podocytes
possess the small GTPase Rab3A, a fine modulator of highly regulated
exocytosis, highly enriched in synapses, which in podocytes associates to
vesicles contained in the foot processes.
rab3a synaptopodin
merge
DAPI
Rastaldi MP, et al. Am J Pathol 2003; 163: 889-899

7. Next, we have shown that Rab3A positive vesicles contain glutamate and are
able to undergo spontaneous and regulated exocytosis/endocytosis with
glutamate release.
Rastaldi MP, et al. FASEB J 2006; 7:976-8

8. Glutamate - mouse GMR7- mouse
Glutamate - human NMDAR1 - human
1 2 3 MWM 4 5 6
100kD
NMDAR1 - mouse
Rastaldi MP, et al. FASEB J. 2006; 20:976-8.

9. To understand the relevance of this glutamatergic system to the
maintenance of the glomerular filter barrier we have decided to study
conditions in which the system was altered, either in the vesicular or in
the receptor compartments.
• Vesicular compartment:
effects of the absence of Rab3A in vitro and in vivo
• Receptor compartment:
effects of blocking the NMDAR1 in vitro and in vivo

10. Vesicular compartment: the Rab3A-KO model
Giardino et al, submitted

11. Vesicular compartment: the Rab3A-KO model
Giardino et al, submitted

12. Vesicular compartment: the Rab3A-KO model
Giardino et al, submitted

13. Vesicular compartment: the Rab3A-KO model
Giardino et al, submitted

14. Receptor compartment: the NMDA glutamate receptor
Ion gradients are widely used in transmembrane signalling.
In neurons, the encoding, transmission, and integration of information relies on the
careful control of transmembrane electrostatic potential by ion channels.
Receptors for glutamate are divided into two broad families: ionotropic (ion
channels) and metabotropic (G-protein coupled) receptors. NMDARs are the most
complex of the ionotropic receptors, and function as calcium channels.
Typically, ligand-gated ion channels
are closed in the resting state, but
open in response to the binding of
agonists, allowing selected ions to
flow down the electrochemical
gradients through an internal pore.
NMDA receptors are composed of multiple subunits, including at least
one NR1 subunit, and one or more modulatory NR2 subunits. NR1
subunit is synthesized to excess, and are retained in the endoplasmic
reticulum until they assemble with NR2.

15. Receptor compartment: the NMDA glutamate receptor
Human kidney Mouse kidney
Podocyte cell line Primary podocyte

16. To understand if glutamate signalling through the NMDAR is important for
podocyte homeostasis, we incubated our cells with a non competitive
antagonist of the ionotropic glutamate receptor NMDAR1 (Norketamine
hydrochloride – 50uM for 30 min), acting by binding to the PCP site located
within the ion channel, thus preventing Ca2+ flux.
In a further series of experiments, we added the agonist NMDA.

17. Giardino et al, submitted

18. Giardino et al, submitted

19. Medium, NMDAR1
Giardino et al, submitted

20. Podo Podo Podo Podo
MWM
control control control norket
NMDAR1
tubulin
Norketamine, NMDAR1
Giardino et al, submitted

21. Glomerular Albumin Permeability Assay
It can be calculated on isolated glomeruli as an index corresponding to the
change of glomerular volume caused by a gradient of albumin
Kidney cortex
sieving
Videomicroscopy and measurement
Substance to
be tested

22. Albumin permeability index, PAlb
The same norketamine
dosage (50uM) and
incubation time (30 min)
we used on podocytes in
Albumin permeability index, PAlb
vitro, were able to modify
albumin permeability in
isolated glomeruli.
Incubation time (min) *P< 0,005 compared to control
Giardino et al, submitted

23. 1
0,9 Norketamine 50uM 30 min, followed by NMDA 100uM 15 min;
Albumin permeability index, PAlb
0,8 ** P< 0,001 compared to Norketamine 50uM
0,7
0,6
----------------------------------------------------------
0,5
0,4
0,3 **
0,2
0,1
0
Ctrl Norketamine + NMDA MK801+ NMDA
Addition of the agonist NMDA after the treatment with the antagonist
restores PAlb index in the normal range.
Giardino et al, submitted

24. In vivo norketamine administration
MWM 2 3 4 MWM 5 6 7
1
a)
75kDa
50kDa
Norketamine hydrochloride (3, 4,
5, 6) or saline (2, 7) were
37kDa
administered (0.3mg/100g) IP
for 3 days to 3 month old mice.
25kDa
b)
saline norketamine
Giardino et al, submitted

25. Norketamine treated

26. Podocin, saline Podocin, norketamine
ZO1, saline ZO1, norketamine
Nephrin, saline Nephrin, norketamine
Giardino et al, submitted

27. NMDA-R1, saline

28. NMDA-R1, norketamine

29. In vivo norketamine administration
1
0,9
0,8
0,7
0,6
P alb value
0,5
0,4
0,3
0,2
0,1
0
Control Norketamine
Glomerular Albumin Permeability Assay conducted on
glomeruli isolated from vehicle and norketamine-injected mice.

30. Human study
Giardino et al, submitted

31. Glomerular signalling hypothesis
Slit diaphragm
Foot processes
neurotransmitter
receptors
Nephrin (Ig-like)
Neph1 (Ig-like)
α3β 1-integrin
dystroglycans
dystroglycans
P-cadherin
β integrin
FAT1 (protocadherin)
Laminin beta2-chain GBM
Agrin
neurotransmitter receptors
Fenestrated endothelium
mesangial cell
Our data seem to support the idea that this system of signaling has a
role in the maintenance of podocyte homeostasis and its dysregulation
can impair the glomerular filter.

32. Rodewald R, Karnovsky MJ. J Cell Biol 60: 423-433, 1974

33. Acknowledgments
Renal Research Laboratory
L. Giardino: animal studies
S. Armelloni: cell culture studies
A. Corbelli: EM and immunogold EM
D. Mattinzoli, M. Ikeahata: histology and immunohistochemistry
M. Li: cell biology
S. Berra: molecular biology
Trieste University
C. Zennaro, M. Carraro: Albumin permeability assay
Rouen University Hospital & INSERM, Paris
D. Guerrot, F. Tourrel: Human study