Peter C. Brazy

Inhibition of Renal Metabolism. Relative effects of arsenate on sodium, phosphate, and glucose transport by the rabbit proximal tubule.

These studies examine the inhibitory effects of arsenate on the transport of sodium, phosphate, glucose, and para-aminohippurate (PAH) as well as oxidative metabolism by proximal convoluted tubules from the rabbit kidney. Transport rates were measured with radioisotopes in isolated and perfused segments. Metabolic activity was monitored through oxygen-consumption rates and HADH fluorescence in parallel studies in suspensions of cortical tubules. The addition of 1mM arsenate to the perfusate reduced fluid absorption rates from 1.24 +/- 0.17 to 0.66 +/- 0.19 nl/nm.min (P < 0.01) and lumen-to-bath phosphate transport from 9.93 +/- 3.47 to 4.25 +/- 1.08 pmol/mm.min (P < 0.01). Similar concentrations of arsenate reduced glucose transport only slightly from 66.1 +/- 6.0 to 56.8 +/-4 4.6 pmol/mm.min (P < 0.05) and had no effect of PAH secretion. Removing phosphate from the perfusate did not affect the net transport of sodium or glucose. In suspensions of tubules, arsenate increased oxygen consumption rates by 20.5 +/- 2.9% and decreased NADH fluorescence by 10.8 +/- 1.5%. These effects on metabolism were concentration dependent and magnified in the presence of ouabain. The data indicate that arsenates main effect is to uncouple oxidative phosphorylation, and that graded uncoupling of oxidative metabolism causes graded reductions in the net transport of both sodium and phosphate. Glucose transport is inhibited only slightly and PAH secretion is not affected. Thus, partial as opposed to complete inhibition of metabolism reveals that different relationships exist between net sodium transport and the transport of phosphate, glucose, and PAH by the proximal renal tubule.

Metabolic Requirement for Inorganic Phosphate by the Rabbit Proximal Tubule: EVIDENCE FOR A CRABTREE EFFECT

These studies examine the effects of acute changes in the availability of inorganic phosphate on the function of isolated proximal renal tubules from rabbit kidney. We removed phosphate from the extracellular fluids and measured fluid absorption rates in isolated perfused tubules and oxygen consumption rates in suspensions of cortical tubules. In proximal convoluted tubules, the selective removal of phosphate from the luminal fluid reduced fluid absorption rates from 1.11+/-0.12 to -0.01+/-0.08 nl/mm . min. This effect on fluid absorption was dependent on the presence of glucose transport and metabolism. The addition of phlorizin to the phosphate-free luminal fluid preserved fluid absorption rates (1.12+/-0.12 nl/mm . min) as did the substitution of nonmetabolized alpha-methyl d-glucopyranoside for glucose (1.05+/-0.21 nl/mm . min) or the addition of 2-deoxyglucose, an inhibitor of glycolysis, to the bathing medium (1.01+/-0.15 nl/mm . min). There was no effect on fluid absorption if phosphate was removed from the bath only. Additionally, removal of phosphate from the luminal fluid of proximal straight rather than convoluted tubules had no effect on fluid absorption rates. Oxygen consumption rates in suspensions of cortical tubules were reduced from 18.9+/-0.6 to 10.6+/-0.6 nmol O(2)/mg tubular protein . min by the removal of phosphate from the medium. This inhibition was prevented by the substitution of alpha-methyl d-glucopyranoside for glucose in the phosphate-free medium. The data indicate that under certain conditions, proximal convoluted tubules require the presence of phosphate in the luminal fluid to preserve tubular function. In the absence of intraluminal phosphate, glucose metabolism causes a reduction in both oxidative metabolism and fluid absorption. This response is analogous to the Crabtree effect and suggests limitations on the intracellular availability of inorganic phosphate.

Phosphate and Glucose Transport in the Proximal Convoluted Tubule: Mutual Dependency on Sodium

The possibility that diverse solutes such as glucose and amino acids may influence phosphate transport in the proximal renal tubule (1) has provided a basis for various theories regarding the mechanism of phosphate absorption. Such theories may include a polyfunctional carrier, a common, limited energy source or shared driving forces. Thus, as with other epithelia (2), absorptive processes in the proximal convoluted tubule may share a common element such as coupling to sodium transport. In this regard, the present studies were designed to examine the relationship between glucose and phosphate absorption in isolated proximal convoluted tubules from the rabbit kidney and to document that sodium transport is necessary for the renal absorption of both these solutes.

Interactions between phosphate transport and oxidative metabolism in the rabbit proximal tubule.

In proximal renal tubules, inorganic phosphate is both a transported solute and a substrate for intracellular metabolism. While these two functions may involve separate pools of phosphate, it is more likely that there is some interaction between phosphate transport and the metabolic processes which use inorganic phosphate. Within the proximal tubular cell, several types of metabolic processes involve inorganic phosphate. Glycolysis and oxidative phosphorylation utilize inorganic phosphate in the production of ATP. On the other hand, gluconeo-genesis and the sodium-potassium ATPase hydrolyze ATP to generate inorganic phosphate. Furthermore, the intracellular concentration of inorganic phosphate may contribute to the phosphorylation potential, i.e. [ATP]/[ADP]·[Pi], which regulates the rates of these metabolic processes.

Intracellular Processes that Affect Renal Phosphate Transport

Studies of phosphate transport in the proximal renal tubule have focused recently on interactions with intracellular processes, especially oxidative metabolism and gluconeogenesis. Intracellular inorganic phosphate is essential for oxidative phosphorylation and participates in mitochondrial respiration. Gluconeogenesis liberates phosphate. This review will summarize our recent observations on changes in phosphate transport with metabolic inhibitors, metabolic substrates and changes in gluconeogenesis.

Energy Compartmentation and Active Transport in Proximal Kidney Tubules

The primary work of the kidney is active transport.1 It is a long-standing observation that a linear relationship exists between the rate of sodium reabsorption by the whole kidney and its rate of oxygen consumption.2,3 Since the oxygen is consumed at the mitochondria and the energy for active transport is used by the Na,K-ATPase located at the plasma membrane on the basolateral side, a basic question in cellular physiology concerns the mechanism whereby the two processes are linked. The answer to this question leads directly to energy compartmentation.

Interactions between Inorganic Phosphate and Energy Metabolism in Renal Cortical Tubules

Inorganic phosphate interacts with essentially all major metabolic pathways, primarily through the phosphorylation of intermediates via ATP or other nucleotides. In this regard, oxidative phosphorylation has an absolute requirement for inorganic phosphate (1) but it is not certain whether phosphate under certain conditions may regulate or control mitochondrial respiration. It is generally believed that mitochondrial respiration is determined by the extramitochondrial concentration ratio of [ADP]/[ATP]. On the other hand, Wilson et al (2) have advanced the concept that mitochondrial respiration may be governed by the extramitochondrial activities of inorganic phosphate as well as ADP and ATP. It is clear that inorganic phosphate is important for mitochondrial anion transport processes that facilitate the transfer of reducing equivalents between cytosolic and mitochondrial compartments (3). In view of these considerations, we performed a series of studies that examine the interactions between phosphate and respiration in renal cortical tubules.

MECHANISMS OF RENAL EPITHELIAL TRANSPORT OF PHOSPHATE

Publisher Summary The combination of isolated perfused renal tubules and isolated membrane vesicles has expanded the information available regarding the renal handling of phosphate. This chapter describes the observations made relative to three areas of interest: segmental localization of phosphate reabsorption, functional heterogeneity for phosphate transport, and cellular mechanisms of phosphate reabsorption. It also focuses on the data obtained using the isolated perfused renal tubule technique and the study of membrane vesicles derived from renal brush border material. Proximal phosphate reabsorption occurs by sodium-dependent interactions between intraluminal phosphate and a brush-border membrane component, and that the mobility of this complex is affected by the electrical and chemical gradients across the luminal membrane. Completion of transepithelial movement of phosphate appears to occur via linkage of metabolically dependent sodium extrusion to the pool of intracellular phosphate that is available for transport.

Phosphate Transport in Isolated and Perfused Renal Tubules

In summary, studies of phosphate transport by isolated renal tubules perfused in vitro confirm many of the data obtained by other techniques with regard to localization. The major contributions of the technique revolve around studies of segments not accessible to micropuncture techniques and around studies of epithelial transport processes that are difficult to examine in vivo.