Address:

Centre de Recherche des Cordeliers, UMRS 1138, ERL 8228

Escalier J, rez-de-chaussée.

15 rue de l'école de médecine

75006 Paris

Contacts:

-Scientific overseers

-Technical overseer

 Gaëlle Brideau    01 44 27 51 12        gaelle.brideau@crc.jussieu.fr

Platform activities:

• Measurements of the glomerular filtration rate and the filtered charge of solutes.
• Determination of urinary ionograms.
• Analysis of water and ion transport and their hormonal regulation in nephron segments.
• Ion transporter analysis and ATPase measurements in nephron segments.
• Gene and protein expression analysis in whole kidneys, nephron segments, or kidney slices.

       1. In vitro microperfusion and micro-fluorescence:

Goals:

Measurements of transport activity in isolated nephron segments.

Measurements of transepithelial voltage.

Measurements of intracellular calcium variations.

Apparatus:

- Two in vitro microperfusion sets are fitted for perfusing and collecting tubules.  Each set consists of an inverted microscope and two micromanipulators mounted on anti-vibration table surrounded by a Farraday cage. Bathing solutions are heated before flowing into the perfusion chamber where the tubule is held. Tubule perfusion rate is modulated thanks to hydrostatic pressure. PDte is measured between the tip of the perfusion pipette and the bath thanks to a millivoltmeter. With this set-up, one can measure water, sodium, chloride, potassium, magnesium,ammonium and calcium net fluxes while the recording transepithelial potentiel difference (PDte). Moreover, one of the two sets is equiped to measure fluorescence at a cellular scale (LED exitation and IXon+ caption system).

- One microforge to make all glass pipettes needed.

One Dionex device (Ultimate 3000) used to measure pmols of Na+, K+, Mg2+ and creatinine.

One chloride titrating device adapted to measure chloride concentrations in pl drops.

One nanoflow to measure Ca2+ and NH4+.

2. Metabolic studies

Goals : Realize urinary ionogrammes

Apparatus:

48 individual mice metabolic cages.

 25 individual rats metabolic cages.

Food and water intake can be controlled. Urine and feces are separated allowing precise measurements of diuresis.

One Konelab 20I and one flame photometer (IL943) allow routine assay. While the flame photometer can dose sodium and potassium in urine and serum samples, the Konelab can measure creatinine, calcium and magnesium, toxins or drogues in different sorts of samples (urine, plasma, serum, LCR, etc…).

We are also equipped with a blood gas analyzer (Epoc) and an osmometer (Vogel 6300).

The blood gas analyzer is used on fresh mice or rat blood to measure sodium, potassium, chloride, carbonic acid and pH. The osmometer is used to determine urine osmolarity.

3. Applications on isolated tubules :

Goals: Genomic and proteomic analyse of renal segments seperated by hand-microdissection.

Apparatus:

7 micro-dissection stations are equipped with binocular loupes and optic fibers to isolate by hand any nephron segment.

1 microscope equipped with a Leitz camera and controlled by a software (Visilog) allowing tubule length measurements.

2 Real time PCR apparatus (LightCycler 480 et LighCycler Carousel-based System) to study gene expression in specific segments.

1 scanner for western blot protein quantification (Licor Odysée) and 1 fluorescence microscope (Axiovert 200M) equipped with 4 filters: dapi, rhodamine, GFP and Cy5, for immunohistochemistry on isolated tubules, allows studying cellular expression and localization of proteins in nephron segments (depending on existing antibodies).

Access conditions:

The material is accessible to all the Cordeliers Research Centre teams  and external teams after consulting the platform overseers.

Contact:

All requests must be adressed to

        Gaëlle Brideau  - email : gaelle.brideau@crc.jussieu.fr

                                    Tél. :  01.44.27.51.12 or 01.44.27.50.03

For inquiries concerning:

a. Integrated Studies:

Projects are discussed with platform overseers to determine their feasibility, resources, and schedules. These projects are conducted in collaboration with one or more of members of the "Edwards/Houillier" team.

b. assays:

• Occasional: the assays are performed by the platform staff.

• Recurrent: training is provided by the platform staff , then the material is freely available.

prices :

Operating costs are not supported by  "Edwards/Houillier" Team, and services are billed to users of the facility.