De novo pyrimidine nucleotide biosynthesis in isolated rat glomeruli

Uracil ribonucleotide-sugars and aminosugars are required for glomerular basement membrane (GBM) biosynthesis. Since these nucleotides are metabolic derivatives of uridine 5'-triphosphate (UTP), we have studied the cellular pools of uridine 5'-diphosphoglucose (UDPG), uridine 5'-diphosphoglucuronic acid (UDPGA), uridine 5'-diphospho-N-acetyl glucosamine (UDPAG) and UTP, and measured UTP synthesis de novo in isolated glomeruli incubated in vitro. Improved techniques for nucleotide quantitation were established and the optimal conditions for glomerular isolation and incubation determined. Substantial quantities of uracil ribonucleotide coenzymes and an active utilization of orotate for the synthesis of pyrimidine nucleotides were demonstrated. UTP synthesis and the pools of UDPG and UDPGA varied markedly with changes in the experimental conditions. The adverse effects of suboptimal conditions were more apparent in glomeruli from diabetic animals than in controls. The use of suboptimal conditions could provide misleading information on GBM metabolism in isolated glomeruli since uracil ribonucleotide coenzyme availability might be reduced.