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* Renal Immunopathology Laboratory, Associazione Nuova Nefrologia, Fondazione D’Amico per la Ricerca sulle Malattie Renali, c/o Department of Nephrology and Immunology, San Carlo Hospital, Milan, Italy;
Microscopy and Image Analysis Consortium, Department of Neurosciences and Biomedical Technologies, Milano Bicocca University, Monza, Italy;
Molecular Medicine, Biomedicum Helsinki, Helsinki, Finland;
Protein Chemistry Unit, Biomedicum Helsinki, Helsinki, Finland;
|| Medical Policlinic, University of Munich, Munich, Germany;
¶ Sanofi-Aventis, Frankfurt, Germany;
# Neurobiology of Learning Unit, Universita’ Vita-Salute S. Raffaele, Segrate, Italy; and
** Biology of Neurodegenerative Diseases Laboratory, Mario Negri Institute, Milan, Italy
1Correspondence: Renal Immunopathology Laboratory, Associazione Nuova Nefrologia and Fondazione D’Amico per la Ricerca sulle Malattie Renali, c/o San Carlo Borromeo Hospital, via Pio II, 3, Milan, 20153, Italy. E-mail mp.rastaldi{at}fastwebnet.it
SPECIFIC AIMS
In the renal glomerulus, podocytes are highly differentiated, ramified cells that have a major role in the maintenance of the filtration barrier. Similarities between podocytes and neurons have been described and our group has previously shown that the small GTPase Rab3A is present in podocytes. Rab3A is a protein involved in processes of highly regulated exocytosis. For this reason, it is most abundant in neurons, where it modulates the vesicle fusion step at the presynaptic membrane. The finding of Rab3A in podocytes has therefore raised the question about its function in these cells, where highly regulated exocytosis was never taken in consideration. Therefore, the first aim of the present work was to get information on Rab3A-interacting proteins in podocytes and have a basis to understand the possible function of Rab3A in these cells.
PRINCIPAL FINDINGS
1. Rab3A extracted from normal glomeruli coimmunoprecipitates with molecules involved in the synaptic vesicle cycle
Rab3A was immunoprecipitated from normal mouse kidney glomeruli and, for comparison, from normal mouse brain. After SDS-PAGE electrophoresis and silver staining, 6 bands selected on the basis of their apparent overexpression in glomerular immunoprecipitates, were trypsin digested and analyzed by mass spectrometry. This analysis did not demonstrate coimmunoprecipitation of Rab3A with any specific podocyte protein. It did, however, show the coimmunoprecipitation of Rab3A with synaptotagmin 1 and glycine glutamate thienyl-cyclohexyl-piperidine binding protein, which are molecules involved in neuronal processes of synaptic transmission.
2. Podocytes possess glutamate-containing vesicular structures that undergo spontaneous and regulated exoendocytosis
Foot processes of podocytes have been described to contain vesicular structures, and we have shown by immunogold electron microscopy (EM) some of these vesicles to be positive for Rab3A.
Indeed, by immunocytochemical analysis we have found that Rab3A-positive vesicles are also strongly positive for glutamate. EM performed on kidney sections and cultured podocytes confirmed the presence of a number of vesicles located in podocyte processes (Fig 1
A, B), and immunogold EM showed glutamate particles contained in these vesicular structures (Fig 1C
). In parallel experiments we tested for the presence of glutamate carriers. This analysis uncovered the presence in podocytes of the vesicular glutamate transporter VGlut1 and of the vacuolar proton pump (V-ATPase), suggesting that podocytes are able to recruit glutamate into vesicles, as it happens in glutamatergic neurons.
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From these results, we hypothesized that the vesicular structures seen in podocyte foot processes might undergo exocytic/endocytic cycling. Therefore, cultured podocytes were incubated with rabbit antisynaptotagmin 1 (anti-N-terminal lumenal domain) antibodies for a 2-h period. On the basis of the occurrence of fluorescent puncta in all podocytes investigated, we concluded that these antibodies were efficiently internalized (Fig 2
A). Furthermore, after fixation, cells were probed with a monoclonal antibody directed against the cytosolic portion of synaptotagmin 1 (Fig 2B
). The precise colocalization of the two markers, definitely demonstrated that staining occurs by specific interaction with synaptic-like vesicles (Fig 2C
). When the same experiment was repeated after incubation with tetanus toxin, which inhibits neurotransmitter release, the uptake of the antilumenal epitope of synaptotagmin (Fig 2E-G
) was abolished.
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To understand the possible regulation of these exoendocytotic processes, we performed further studies using alpha-latrotoxin, a presynaptic neurotoxin widely applied to trigger neurotransmitter release from synaptic vesicles.
Applying subnanomolar (0.5 nM) and nanomolar (2.5 nM) 
-latrotoxin concentrations on cultured podocytes, we found that glutamate release was actually taking place with kinetics that precisely parallel those described in neurons.
3. Microarray expression data show that glomeruli express a wide range of neuron-specific molecules
To get a global view of the molecules involved in synaptic transmission that are possibly synthesized by normal human glomeruli, we generated microarray expression data from microdissected glomeruli of transplant living donor biopsies. These data have shown that normal human glomeruli do transcribe a complete series of synaptic molecules, neurotransmitter receptors, and neuron-specific molecules.
CONCLUSIONS AND SIGNIFICANCE
Glomerular podocytes are highly differentiated cells with a complex ramified structure: few major processes give rise to many fine pedicels or foot processes, which interdigitate with corresponding projections of neighbor cells (Fig 3
). Specialized adhesions are interposed among foot processes, forming the so-called slit diaphragm, and between the basal domain of foot processes and the glomerular basement membrane. It has been recently described that both the slit diaphragm and the basal domain are highly dynamic signaling domains, and our present results strongly suggest that signals can be triggered in a synaptic-like way.
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Our investigation shows, in fact, that podocytes possess vesicular structures that strictly resemble synaptic vesicles; they are composed of synaptic vesicle molecules, are able to undergo spontaneous and regulated exocytosis/endocytosis and release glutamate.
Cells need to talk to each other, and more differentiated, highly specialized cells have developed sophisticated means of communication that reach the highest expression in neuronal cells, but are not limited to them. The concept of immunological synapse provides a very good example, and the functional expression of glutamate-signaling molecules has been described for several nonneuronal cell types, mainly but not only belonging to the endocrine system.
It is our opinion that, given the complexity of podocyte structure and the continuous stimulation and stress by blood pressure and contents, these cells are likely in need of a precise and fast modality of communication among themselves and with the other glomerular cells.
Furthermore, our data seem to prove that normal glomeruli express a wide array of neuron-specific molecules, and recent data have actually shown that a depolarization, due to increase of free intracellular calcium, does occur by exposure of podocytes to dopamine and acetylcholine via specific receptors, suggesting for podocytes an even more neuron-like behavior.
On the basis of this view, the slit diaphragm should be regarded as a synaptic adhesion, composed by synaptic adhesion molecules. Considered in this way, many already described features fall into place, starting from the morphological aspect of the slit diaphragm, which is a modified adherens junction where staggered cross-bridges extend from the slit walls to a longitudinal central filament, generating its typical zipper-like structure. The molecular composition of the slit diaphragm also fits well with a synaptic adhesion. It is known that neuron-neuron synapses bear at least cadherin-like and immunoglobulin (Ig) superfamily-like adhesion molecules. The slit diaphragm is precisely composed of both Ig-like molecules, such as nephrin and NEPH1, and cadherin molecules, such as P-cadherin, and the proto-cadherin FAT.
As for their function, apart from promoting the stability of synapses, many data support the role of synaptic adhesion molecules in target recognition, that is, to help in choosing the right partners from a network of processes. Establishing interdigitations, differentiating podocyte foot processes possibly use the same mechanism to find their right place and partner, and in this respect, it seems worth noting that SYG-1, the C. elegans ortholog of NEPH-1, is a molecule that has been isolated in a genetic screen for mutants defective in synaptic positioning.
Our "synaptic view" also fits with the adhesive properties of the basal cell membrane of foot processes with the glomerular basement membrane. In this case, a kind of neuromuscular junction comes to mind, first because of the presence of a basal lamina, second because of its composition by integrins, dystroglycans, and especially agrin, considered to be a critical nerve-derived organizer of postsynaptic differentiation in neuromuscular junctions. Furthermore, the so-called synaptic laminin (or laminin ßbeta;2 chain) is selectively expressed by the synaptic basement membrane of neuromuscular junctions and by the kidney glomerular basement membrane.
In conclusion, we believe that our hypothesis of synaptic transmission in podocytes may help to better explain several already known glomerular physiological features and surely offers a different perspective for understanding glomerular cell signaling that may be important for advancing of knowledge of glomerular cell behavior. This is especially needed because chronic renal failure is increasing worldwide, whereas there are few therapeutic options, and those that are mainly available consist of generic immunosuppressive drugs or symptomatic treatments, due to the still limited knowledge of kidney cell biology.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4962fje
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