Glutamate dehydrogenase activities in microdissected rat nephron segments: effects of acid-base loading

Abstract

A method for measurement of glutamate dehydrogenase (GDH) activity in single renal tubules was employed to determine the distribution and regulation of GDH in tubule segments. Fresh microdissected tubules from collagenase-treated kidneys were permeabilized by hyposmotic shock and freezing. The rate of conversion of alpha-ketoglutarate, NH4+, and NADH to glutamate and NAD was measured at 37 degrees C fluorometrically. Very high activities were found in proximal tubule segments (150-210 pmol.min-1.mm tubule length-1), intermediate values (40-90 pmol.min-1.mm-1) in distal convoluted tubules, cortical thick ascending limbs, connecting tubules, medullary thick ascending limbs, and lower values (5-30 pmol.min-1.mm-1) in cortical collecting ducts, inner medullary collecting ducts, outer medullary collecting ducts, outer medullary thin limbs, and inner medullary thin limbs. To determine the effects of acid-base loading on GDH activity, 0.28 M NH4Cl (acid) or 0.28 M NaHCO3 (alkali) was added to the animals' drinking water for 7 days. Acid intake by the rats increased GDH activity in S1 and S2 proximal tubules by threefold, with no effect in other segments, including S3 proximal tubules. Alkali intake decreased GDH activity in the S3 proximal tubule by 40%, with no effect in other segments. We conclude that GDH activities are highest in proximal tubule segments and are regulated only in proximal tubule segments. Thus the results are consistent with the view that the proximal tubule is the chief site of the regulated production of ammonium in the kidney.