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Abstract: TH-PO486

Functional Proteomics of Isolated Perfused Cortical Collecting Ducts

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Himmerkus, Nina, Christian-Albrechts-University Kiel, Kiel, Germany
  • Bleich, Markus, Christian-Albrechts-University Kiel, Kiel, Germany
  • Benzing, Thomas, University Hospital Cologne, Köln, Germany
  • Rinschen, Markus M., University Hospital Cologne, Köln, Germany
Background

In renal physiology and pathophysiology large-scale omic data sets are generally generated from bulk samples, e.g. the complete kidney or cortex vs. medulla. It is therefore difficult to predict implications for functional changes taking place in the individual, highly specialized and very distinct nephron segments. In the context of hypertension it is of high clinical relevance to understand regulation of salt transport in the collecting duct and the phenomenon of salt-sensitivity. The aim of this study was to establish a method to correlate measured electrophysiological properties of individual, isolated perfused cortical collecting ducts (CCD) with respective proteomic data.

Methods

For the electrophysiological properties, freshly isolated CCDs of 6-8 week old C57Bl6 mice were investigated in a double-barreled perfusion system. Transepithelial voltage (Vte) was recorded and transepithelial resistance as well as amiloride sensitive equivalent short-circuit current (ΔI’sc) were calculated. After these measurements every single tubule was recovered, processed and ultrasensitive mass spectrometry was performed.

Results

In manually collected CCD segments we were able to identify more than 4000 distinct proteins. In the perfusion experiments, protein expression patterns typical for intercalated cells or, principal cells, respectively, showed negative correlation in the proteome. Vte varied between -8.5 and -32.5 mV and correlated with the regulator protein NEDD4. ΔI’sc as a measure of electrogenic sodium reabsorption was reflected by the expression level of the b-subunit of the epithelial sodium channel.

Conclusion

We could show that functional proteomics on a single nephron segment is possible and allows function-proteome correlations. This unique data acquisition approach will allow conclusions on physiological or pathophysiological outcomes based on omics data at the level of functional units rather than total organ samples.