Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB134

Poster Communications

Correlation of proteomics and function in isolated perfused single cortical collecting ducts

N. Himmerkus1, M. Bleich1, T. Benzing2, M. Rinschen2,3

1. Institute of Physiology, Christian-Albrechts-University Kiel, Kiel, Germany. 2. Department II of Internal Medicine, University Hospital Cologne, Köln, Germany. 3. Center of Metabolomics and Mass Spectrometry, The Scripps Research Institute, San Diego, California, United States.

The understanding of the kidney‘s ability to regulate and maintain salt and water homeostasis is highly relevant in modern medicine: by this fine-tuning the kidney keeps blood volume (i.e. blood pressure) and plasma electrolyte composition in a narrow range. Pathological misbalancing easily leads to hypertension or endangers excitable tissues like the heart. On the other hand the kidney is action side of powerful pharmacotherapy to correct misbalancing. Especially with respect to hypertension the distal parts of the nephron, its salt-sensitivity and regulation have been in the focus of investigation. Cortical collecting ducts (ccd) of 6-8 week old C57Bl6 mice have been mechanically isolated and immediately investigated electrophysiologically in a double barreled perfusion system. The length of the tubule was taken as a measure of protein amount. Transepithelial voltage (Vte), transepithelial resistance and amiloride sensitive equivalent short-circuit current (ΔI'scamil) were measured to estimate the functional state of the tubules. For every single tubule ultrasensitive mass spectrometry was then used to determine the expression level of a distinct set of tubular proteins. CCDs were of 400-600 µm of length, showed lumen negative Vte between -8.5 and -32.5 mV and an ΔI'scamil between 54 and 192 µA/cm2. The tubular length was correlated with actin and Na+-K+-ATPase expression. Vte correlated with the regulator protein NEDD4 and, intriguingly, the water channel AQP2. ΔI'scamil reflected the expression level of the b-subunit of the epithelial sodium channel. In contrast to large-scale omic data sets generated from bulk samples which suffer from crude biochemical isolation procedures, single-tubule functional proteomics predicts physiological function of the respective individual nephron segment. It allows identification of novel players within regulatory networks and a better understanding of how different cell types and transport systems in this segment are interconnected and regulated in a concerted manner.

Where applicable, experiments conform with Society ethical requirements