Application of cold flush preservation to in vitro microperfusion studies of kidney tubules

Abstract

The technique of microperfusion in vitro of isolated segments of the nephron, first described by Burg eta! [1], has led to major advances in our understanding of nephron function. The technique has been applied to all segments of the nephron, many of which are inaccessible to other forms of study (see Kidney mt 22(5), 1982). Two limiting factors that have influenced the application of this technique are: (1) the ease with which a tubule segment can be dissected from kidney tissue and (2) the viability of the tubule segment during perfusion, particularly when proximal segments are perfused. Many laboratories have limited their observations to New Zealand white rabbits, which possess kidneys that are more readily dissectable than those of other mammals; even so, the occurrence of readily dissectable kidneys varies considerably. Kidney tissue is customarily cooled for dissection to minimize the development of ischemic tubule damage [21; nevertheless, it is common practice to use another rabbit if the dissection time exceeds 30 to 60 mm. We have recently made use of a technique that permits extension of the dissection time and provides several other practical advantages. The technique involves the application of a modified cold preservation procedure similar to that used in kidney transplantation. It arises from a new type of cold flush solution which consists of a phosphate buffer with added sucrose (PBSuc) shown to provide excellent preservation of tubule morphology in rat kidneys [3]. Our observations suggest that it also offers effective preservation of function as judged by measurements of fluid reabsorption rate (Jv), transmural potential difference (PD), leak of iothalamate from lumen to bath, and the responses to removal of intraluminal glucose and alanine.