Nephrotoxicity assessment by measuring cellular ATP content: I. Substrate specificities in the maintenance of ATP content in isolated rat nephron segments

Abstract

To clarify the characteristics of cellular ATP synthesis in individual nephron segments for assessing nephrotoxicity of chemicals, cellular ATP content was measured by the luciferin/luciferase system under various conditions using intact nephron segments isolated from male Sprague-Dawley rats. Increasing the duration of collagenase treatment of kidney slices significantly lowered the cellular level of ATP newly synthesized from 2 m m glutamine in PST at 37°C over 30 min ( p < 0.01). The tubular incubation time significantly affected the cellular ATP content in the early and middle portions (S 2) of the proximal tubule ( p < 0.05 and p < 0.01, respectively) over 20 min and in the late proximal tubule over 10 min. Among numerous substrates tested, such as d-glucose, glutamine, pyruvate, dl-lactate, and β-hydroxybutyrate, the substrate utilization for maintaining cellular ATP content was entirely variable according to each nephron segment. Pyruvate and glutamine were the best substrates in the proximal tubule. On the other hand, ATP production from glutamine was less than that from the other substrates in the distally located nephron segments: medullary and cortical thick ascending limbs of Henle's loop (MAL and CAL, respectively), distal tubule, cortical and medullary collecting tubules (CCT and MCT, respectively). In general, glucose, pyruvate, and lactate appear to be equivalent in maintaining ATP content in the distal segments of renal tubules. A monovalent cation ionophore, monensin, at 10 μg/ml decreased the cellular ATP content in MAL, CAL, and MCT significantly. Mercuric chloride (HgCl 2) was used as a model compound to study nephrotoxicity by investigating its effects on cellular ATP metabolism in microdissected nephron segments. HgCl 2 at 1 × 10 −6 m significantly decreased ATP content only in S 2 ( p < 0.05), clearly demonstrating S 2 to be the most sensitive segment within the nephron. These results indicate that measurement of cellular ATP content would be a useful method for forecasting the intrarenal toxic site and potency of possible nephrotoxic chemical compounds.