New Vibro‐Dissociation Method for Isolation of Defined Nephron Segments and Small Renal Vessels
Authors: Isaeva Elena , Fedoriuk Mykhailo , Bohovyk Ruslan , Levchenko Vladislav , Palygin Oleg , Staruschenko Alexander 
Published: June 2021

The assessment of many aspects of nephron and kidney function often requires careful isolation of specific kidney segments. Several different approaches were developed to isolate nephron fragments for downstream analyses in vitro. Mechanical dissection of nephron segments and enzymatic perfusion followed by tissue centrifugation are among the most widely used approaches for electrophysiological, biochemical, molecular and fluorescence microscopy studies in renal physiology. However, these methods have considerable limitations. Mechanical dissection of individual segments is time consuming and provides only a small amount of useful tissue. Prolonged enzymatic exposure as well as centrifugation of renal tissue, could potentially impact cellular viability and properties of membrane proteins. Here we developed an alternative, simple and reproducible method enabling rapid isolation of well‐preserved glomeruli, nephron segments, and small renal vessels. The process is based on the vibro‐dissociation technique, which was first introduced by Vorobjev (J Neurosci Methods, 1991) and quickly became the gold standard for single cell isolation in neuroscience field of research. Similar to brain tissue, mice kidney slices of controlled thickness (0.2–1.0 mm) are prepared using a microtome (EMS OTS‐4000). Slices can be used immediately after cutting or preserved for several hours in a chamber with a mesh bottom for optimal carbogen‐saturated solution exchange. For vibro‐dissociation, local mechanical vibration (GFG‐8250A, function generator frequency 100–150 Hz; amplitude 20–30 μm) with a flame‐sealed ball‐shaped micropipette (size 0.2–0.6 mm) is directly applied to the kidney slice within a few minutes. Importantly, isolation of kidney segments may be performed in predetermined regions such as renal cysts, cortex, or medullary regions, which improves targeted isolation. Our experiments revealed that this approach could be applied with or without enzymatic pretreatment of slices; in the case of enzymatic digestion, short pretreatment for 5–10 min was sufficient for tubules dissociation. Employing this technique, we were able to rapidly separate a substantial fraction of freshly isolated nephron segments, including proximal tubules, thick ascending limb, distal convoluted tubules, and various parts of the collecting duct. In addition, numerous small renal vessels and decapsulated glomeruli or glomeruli containing intact afferent/efferent arterioles were obtained using this approach. The integrity of isolated nephron segments was confirmed with electrophysiological analysis and fluorescence microscopy and purity tested with known markers for specific cell types. This approach has several advantages over existing methods such as simplicity, long term storage of isolated kidney slices, lack or minimal enzymatic treatment, bulk quantities, and pure fraction. Therefore, applying vibro‐dissociation technique for separation of different nephron segments and small renal vessels may be a powerful tool for assessment of their properties in vitro.Support or Funding InformationNIH HL135749 and HL116264; AHA 17SDG33660149This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.