Perfusion of Isolated Kidney Tubules

After its invention by Burg et al. (1966) this technique has been used successfully in the kidney tubule segments of several species man, rabbit, rat, mouse, hamster, snake, birds etc. The tubule segments are dissected from thin kidney slices ( 1 mm thickness). Usually dissection can be done using sharpened forceps or needles without the addition of proteases (collagenase). The segment is identified by its anatom- 

For smaller species, proximal tubule segments can also be isolated from the entire kidney using in situ col-lagenase perfusion techniques (Tyson et al. 1990). Following anesthesia, the kidneys may be perfused via the aorta (mice) or renal arteries (rats, rabbits) for five minutes to remove the residual blood the kidneys and associated blood vessels are then removed and the perfusion continued for an additional 15-20 minutes with buffer containing 180 U/ml of Type I collagenase. The cortical tissue is then removed from the medulla and the tubules isolated as described above. The viability and function of tubules so isolated are comparable to those isolated without collagenase (Rodeheaver et al. 1990). 

Flux measurements (Schafer et al. 1974). The collection rate (Vc, nl/min) can be measured by the constant bore collection pipette by timed collections. Radioactive tracers can be added to the lumen or bath fluid. For instance, radioactively labelled inulin can be added to the perfusate (Inp) and can be used to measure volume absorption DV = perfusion rate (Vj-Vc). Unidirectional fluxes, bath to lumen and lumen to bath, for any given substance can be quantified, and permeabilities (Px) can be determined  

Patch clamp studies. The combination of in vitro perfusion of renal tubules and patch clamp analysis of ion channels in the luminal and basolateral membranes is described in Sect.C. 1.1.4. 

Fluorescent dyes in the isolated perfused tubule. Several fluorescent dyes for the monitoring of Na+, K+, Cl, Ca2+, pH have become available during the past few years. These dyes can be used in the in vitro perfused tubule (Nitschke et al. 1991). The inverted microscope is equipped with an appropriate illumination and filter wheel for excitation. The emission is measured by photon counting or by a video camera. When compared with impalement methods, these techniques are probably easier for routine use. 

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