Origins of Isolated Tubule Microperfusion Methodology

Understanding of renal function has been facilitated by the technique of perfusion of isolated renal tubule segments in vitro. The basic technology originated in the Laboratory of Kidney and Electrolyte Metabolism of the National Institutes of Health in the early 1960s and then was expanded to apply a variety of analytical methods to single tubules.

Possible role of calcium in parathyroid hormone actions in rabbit renal proximal tubules

Using a glucose microassay and in vitro isolated renal tubule perfusion technique, we have studied the actions of parathyroid hormone (PTH) on gluconeogenesis (GNG) and fluid (Jv) and phosphate (Jp) transport rates in isolated rabbit renal proximal tubules. In proximal straight tubules (PST), PTH stimulated GNG and inhibited Jv and Jp. In proximal convoluted tubules (PCT), PTH inhibited Jv but failed to affect GNG and Jp. An increase in Ca concentration, however, stimulated GNG and allowed PTH to inhibit Jp in PCT. Addition of the intracellular Ca antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) abolished the inhibitory effects of PTH on Jv and Jp in both PCT and PST. In conclusion, these studies suggest that Ca-dependent intracellular pathways may be involved in the actions of PTH in rabbit renal proximal tubules. The altered response to PTH in rabbit PCT may be due to alterations in the response of intracellular Ca to the hormone.

The inhibition of parathyroid-hormone actions on gluconeogenesis and phosphate transport by 3-mercaptopicolinate in rabbit renal proximal tubules

By using a glucose microassay and the technique for isolated renal-tubule perfusion in vitro, the addition of 3-mercaptopicolinate, a gluconeogenesis inhibitor which inhibits phosphoenolpyruvate carboxykinase specifically, was found to abolish the effects of parathyroid hormone on gluconeogenesis and phosphate-transport rate in isolated rabbit renal proximal straight tubules, suggesting that these parathyroid-hormone actions may share some unknown, yet 3-mercaptopicolinate-inhibitable, intracellular processes.

Micromethodology for measuring ATPase activity in renal tubules: mineralocorticoid influence

The ATPase activity of rabbit isolated renal tubule segments was measured using a microtechnique in which the hydrolysis of ATP was enzymatically coupled to the appearance of an alkali-converted, highly fluorescent form of nicotinamide adenine dinucleotide. The methods are simple, reproducible, and have a high sensitivity in which picomole quantities of hydrolyzed ATP can readily be measured. Several methods for permeabilizing the cell membranes for measurement of Na+-K+-ATPase activity were evaluated, including osmotic (distilled water or 300 mM imidazole) and temperature (freezing) shock and addition of the nonionic detergent octylglucoside. An octylglucoside concentration of 0.5% was found to cause a maximum activation of the Na+-K+-ATPase and was comparable with that observed when tubules were permeabilized by exposure to distilled water and freezing. Incubation of tubules in 300 mM imidazole was less effective in permeabilizing the cell membranes. In all subsequent studies, the cells were permeabilized by exposure to distilled water and freezing as done by others. The methods were used to assay for the basal levels of Na+-K+-ATPase in the superficial proximal convoluted tubule, the superficial proximal straight tubule, and the cortical collecting tubule and were found to average 44.9 +/- 6.3, 26.4 +/- 2.4, and 11.8 +/- 2.2 pmol ADP X mm-1 X min-1, respectively. Furthermore, elevation of plasma mineralocorticoids by daily injections of deoxycorticosterone acetate (2 mg X kg-1 X day-1) for 4-15 days caused a doubling in the Na+-K+-ATPase activity of the cortical collecting duct, confirming the results of others. The methods presented can easily be adapted for microanalysis of other ATPases.

Ontogeny of glycine transport in isolated rat renal tubules

Isolated renal tubule preparations were made from newborn Sprague-Dawley rats and used to study initial entry rate kinetics of glycine. The results were compared to those obtained in the isolated tubule preparation from the adult rat kidney. While initial rates of glycine uptake were identical for newborn and adult tubules, significant differences in influx kinetics were demonstrated. Of the two apparent transport Km systems shown to be present in the newborn tubule, the high-affinity, low-capacity system accounts for about 40% of total glycine uptake at physiologic concentrations. The high-affinity, low-capacity system of the adult tissue accounts for about 10% of total uptake at the same concentration range. The data lend strength to the argument against the concept that the physiologic hyperglycinuria of the newborn rat is due to either impaired ability to concentrate glycine intracellularly or to absence of one or more transport mechanisms for glycine.