Biochemical Characteristics of The Na-alpha-Ketoglutarate Cotransport System in Proximal Convoluted and Straight Tubules of the Rabbit Kidney

Ahn, Do Whan; Kim, Kyoung Ryong; Park, Hee Seok; Park, Yang Saeng

Abstract

PURPOSE: alpha-Ketoglutarate (alphaKG), a Krebs cycle intermediate, is extensively used in the kidney as a fuel substrate and as a counter anion for organic acid secretion. It is known to be taken up by the proximal tubule cells via the brush-border as well as basolateral membranes. We explored biochemical characteristics of the brush-border and basolateral alphaKG transport systems in pars convoluta and pars recta of the proximal tubule, respectively.
METHODS
Brush-border and basolateral membrane vesicles (BBMV and BLMV) were isolated from rabbit renal outer cortex and outer medulla by Percoll gradient centrifugation. Vesicular uptake of alphaKG was determined by rapid Millipore filtration method using alpha-14[C]KG as a substrate.
RESULTS
Both BBMV and BLMV showed a Na-gradient dependent uphill transport of alphaKG. The systems in both membranes were similarly inhibited by Li and activated by Na (Hill coefficient of 1.4). Kinetic analyses indicated that the Na-alphaKG cotransporters in the BBMV had a lower substrate affinity as compared with those in the BLMV. The transport systems in BLMVs showed a similar Km but different Vmax between the outer cortex (Km: 34 uM, Vmax: 3.3 nmol/mg protein/10s) and outer medulla (Km: 37, Vmax: 1.8). On the other hand, the systems in BBMVs were different in both Km and Vmax between the outer cortex (Km: 194, Vmax: 3.3) and outer medulla (Km: 89, Vmax: 1.7).
CONCLUSION
The findings suggest that both axial and apical to basolateral heterogeneity of the Na-alphaKG cotransport system in proximal tubules may be due to a physiological adaptation to efficiently utilize alphaKG in the kidney.