Purification of rat papillary collecting duct cells: functional and metabolic assessment

Abstract

Rat renal papillary collecting duct (PCD) cells were isolated using collagenase and hyaluronidase digestion and a three-step low-speed centrifugation. As assessed by binding of the lectin Dolichos biflorus and determination of vasopressin-sensitive adenylate cyclase and Na+-K+-ATPase, the enrichment of PCD cells over a crude papillary cell preparation was 1.8, 2.4, and 1.4, respectively. Microscopic evaluation indicated that the preparation was greater than 90% pure PCD cells. The isolated cells were viable as evident from the high K/Na ratio of intracellular electrolytes measured by electron probe analysis (5.3), from the high ATP/ADP ratio (2.15), and the metabolic response to alterations in Na transport. Exposure to 2 mM ouabain or removal of Na reduced O2 consumption by 25-35%; the uncoupler carboxylcyanide-m-chlorophenylhydrazone more than doubled O2 consumption. In the presence of 14 mM glucose and at a PO2 of 100 Torr the cells produced substantial quantities of lactate. This aerobic glycolysis may account for greater than 20% of the ATP production. In the presence of rotenone, glycolysis increased by 56% and was able to maintain the cellular ATP level at 65% of control. In the absence of any exogenous substrate PCD cells respired normally and had a close to normal ATP content, but lactate production was markedly decreased. These results demonstrate that viable PCD cells can be isolated from rat kidney. At normal PO2 and in the presence of D-glucose the cells show a substantial amount of aerobic glycolysis, although their mitochondrial respiration is not rate limiting. In the absence of glucose the cells derive the majority of their energy from an as yet unidentified endogenous substrate.