Vibrodissociation method for isolation of defined nephron segments from human and rodent kidneys

Abstract

Our current knowledge of the properties of renal ion channels responsible for electrolytes and cell energy homeostasis mainly relies on rodent studies. However, it has not been established yet to what extent their characteristics can be generalized to those of humans. The present study was designed to develop a standardized protocol for the isolation of well-preserved glomeruli and renal tubules from rodent and human kidneys and to assess the functional suitability of the obtained materials for physiological studies. Separation of nephron segments from human and rodent kidneys was achieved using a novel vibrodissociation technique. The integrity of isolated renal tubules and glomeruli was probed via electrophysiological analysis and fluorescence microscopy, and the purity of the collected fractions was confirmed using quantitative RT-PCR with gene markers for specific cell types. The developed approach allows rapid isolation of well-preserved renal tubules and glomeruli from human and rodent kidneys amenable for electrophysiological, Ca2+ imaging, and omics studies. Analysis of the basic electrophysiological parameters of major K+ and Na+ channels expressed in human cortical collecting ducts revealed that they exhibited similar biophysical properties as previously reported in rodent studies. Using vibrodissociation for nephron segment isolation has several advantages over existing techniques: it is less labor intensive, requires little to no enzymatic treatment, and produces large quantities of well-preserved experimental material in pure fractions. Applying this method for the separation of nephron segments from human and rodent kidneys may be a powerful tool for the indepth assessment of kidney function in health and disease.

NOX4‐dependent regulation of ENaC in hypertension and diabetic kidney disease

“…Renal tubules were isolated manually or using a vibrodissociation technique described previously. 30,31 Recordings have been made using extracellular solution (in mM): 150 NaCl, 2 MgCl 2 , 10 HEPES (pH 7.4). Patch pipettes were filled with a solution of the following composition (in mM): 140 LiCl, 2 MgCl 2 , and 10 HEPES (pH 7.35).…”

Role of opioid signaling in kidney damage during the development of salt-induced hypertension

“…Thus, overstimulation of κ-ORs during chronic opioid exposure may result in disorganized F-actin arrangement and foot processes effacement in podocytes. In addition, we were able to confirm the presence of κ-OR/TRPC6 signaling in podocytes of freshly isolated human glomeruli, using a recently developed protocol for the isolation of nephron segments and glomeruli from discarded human kidney transplants (Isaeva et al, 2019).…”

“…Podocytes from freshly isolated human glomeruli express κ-ORs and exhibit κ-OR/TRPC6-mediated calcium influx Glomeruli were isolated from discarded human kidney transplants utilizing recently described by us the vibrodissociation method, which further allows assessment of the functional cell properties by confocal or electrophysiological techniques (Isaeva et al, 2019). The human glomeruli were used to delineate the functional role of the κ-OR/TRPC6 pathway in the opioid-mediated calcium response.…”

“…Human kidneys were obtained from the organ procurement organization, perfused with preservation solution, and stored on ice before the glomeruli isolation. Our previous studies revealed that these kidneys are viable and functional in vitro (Isaeva et al, 2019). Glomeruli were isolated by the vibrodissociation or sieving methods, as described previously (Ilatovskaya et al, 2018;Spires et al, 2018;Isaeva et al, 2019).…”

Scanning ion conductance microscopy of live human glomerulus

“…12 Renal glomeruli were isolated using a vibrodissociation technique as previously described. 13 This approach allows the rapid isolation of well-preserved renal glomeruli from human kidneys. To perform SICM imaging, freshly isolated human glomerulus were attached to poly-L-lysine glass surface placed into the cell chamber filled with PSS solution.…”