Fazl Mohyuddin

Localization of the membrane defect in transepithelial transport of taurine by parallel studies in vivo and in vitro in hypertaurinuric mice.

We investigated the mechanism of taurinuria in three inbred strains of mice: A/J, a normal taurine excretor (taut+); and two hypertaurinuric (taut-) strains, C57BL/6J and PRO/Re. Plasma taurine is comparable in the three strains (approximately 0.5 mM), but taurinuria is 10-fold greater in taut- animals. Fractional reabsorption of taurine is 0.967 +/- 0.013 (mean +/- SD) in A/J); and 0.839 +/- 0.08 and 0.787 +/- 0.05 in C57BL/6J and PRO/Re, respectively. Taurine concentration in renal cortex intracellular fluid (free of urine contamination) is similar in the three strains. Taurine reabsorption is inhibited by beta-alanine, in taut+ and taut- strains. These in vivo findings reveal residual taurine transport activity in the taut- phenotype and no evidence for impaired efflux at basilar membranes as the cause of impaired taurine reabsorption. Cortex slices provide information about uptake of amino acids at the antiluminal membrane. Taurine behaves as an inert metabolite in mouse kidney cortex slices. Taurine uptake by slices is active and, at less than 1 mM, is greater than normal in taut- slices. Concentration-dependent uptake studies reveal more than one taurine carrier in taut+ and taut- strains. The apparent Km values for uptake below 1 mM are different in taut- and taut+ slices (approximately 0.2 mM and approximately 0.7 mM, respectively); the apparent Km values above 1 mM taurine are similar in taut+ and taut- slices. Efflux from slices in all strains in the same (0.0105-0.0113 mumol-min-1-g-1 wet wt), but taut- tissue retains about 10% more radioactivity over the period of efflux. beta-Alanine is actively metabolized in mouse kidney. Its uptake in the presence of blocked transamination, is greater; its intracellular oxidation is attenuated; and its exchange with intracellular taurine is diminished in taut- slices. These findings indicate impaired beta-amino acid permeation on a low-Km uptake system at the luminal membrane in the taut- phenotype. beta-Amino acids are not reclaimed efficiently either from the innermost luminal pool in cortex slices or from the ultrafiltrate in the tubule lumen in vivo. The former leads to high uptake ratios in vitro, the latter to high clearance rates in vivo. In vitro and in vivo data are thus concordant. This is the first time that a hereditary defect in amino acid transport has been assigned to a specific membrane surface in mammalian kidney.

A γ-aminobutyrate pathway in mammalian kidney cortex☆

Abstract Rat kidney cortex converts l -glutamate to γ-aminobutyrate by a decarboxylation reaction which differs from the corresponding reaction in brain. Renal l -glutamate decarboxylase has two apparent K m values for glutamate in homogenates (0.4 and 2.5 mM). γ-Aminobutyrate is converted by a transaminase whose capacity appears to exceed the decarboxylase. γ-Aminobutyrate is converted ultimately to succinate and CO 2 . γ-Aminobutyrate stimulates respiration of kidney cortex slices in vitro and the compound crosses cell membranes in kidney by a respiration-linked, mediated process. Chronic acidosis lowers renal γ-aminobutyrate in the rat; brain γ-aminobutyrate is unaffected by acidosis. Glutamic acid decarboxylase and γ-aminobutyrate transaminase activities are unchanged in acidosis. α-Methylglutamate, an inhibitor of renal glutaminase, lowers the γ-aminobutyrate and glutamate content of rat kidney in normal and acidotic states. Aminooxyacetic acid in vivo , an inhibitor of γ-aminobutyrate transaminase, causes a striking increase in renal γ-aminobutyrate during chronic acidosis. At concentrations of glutamate in vitro , which are similar to the tissue glutamate content in vivo , the γ-aminobutyrate pathway accounts for approximately one-fourth of glutamate disposal in rat kidney cortex slices.

The ontogeny of amino acid transport in rat kidney I. Effect on distribution ratios and intracellular metabolism of proline and glycine

Abstract Newborn Long-Evans rat pups have reduced tubular reabsorption of proline, hydroxyproline and glycine in vivo. Reabsorption of iminoacids improves one week after birth; glycine transport improves in the third week. Kidney in vivo accumulates proline and glycine from plasma and urine against a large chemical gradient. Newborn kidney cortex slices in vitro take up l -proline, glycine and α-aminoisobutyric acid less efficiently than mature tissue. The age-dependent differences are most apparent after short incubation ( min ) and at specific concentrations of the substrates. Net uptake of amino acids, after long incubation, is usually greater in newborn kidney than in mature tissue. After uptake proline and glycine incorporation is greater, whereas oxidation and metabolic conversion is less in newborn tissue than in mature kidney. Newborn and adult kidney slices exhibit similar pH optima for l -proline and α-aminoisobutyric acid uptake. Temperature elevation increases their steady-state uptake more in newborn than in adult kidney.

The ontogeny of amino acid transport in rat kidney II. Kinetics of uptake and effect of anoxia

Abstract Low -K m transport of l -proline and glycine and high -K m transport of α-aminoisobutyric acid is absent in newborn Long-Evans rat kidney cortex slices. Other modes of uptake found in mature kidney are present in postnatal kidney. The deficient proline transport appears at one week of age while the systems for glycine and α-aminoisobutyric acid become active by the 3rd week. The ν max of systems present at birth increases 2- or 3-fold during maturation indicating that ontogeny affects both total and specific activity of membrane transport systems in kidney. The findings in vitro correlate with postnatal physiological transport in vivo . Steady-state accumulation of amino acids in vitro is greater in newborn kidney than in mature tissue; reduced efflux accounts for this. Elevation of temperature stimulates efflux less in newborn kidney. Anoxia primarily inhibits low -K m transport of l -proline andglycine. Transport in newborn kidney by the high -K m system is relatively protectedfrom anoxia.

Transport and metabolism of sarcosine in hypersarcosinemic and normal phenotypes

An adolescent male proband with hypersarcosinemia was discovered incidentally in a French-Canadian family; no specific disease was associated with the trait. The hypersarcosinemia is not diminished by dietary folic acid even in pharmacologic doses (30 mg/day). The normal absence of sarcosine dehydrogenase in cultured human skin fibroblasts and in leukocytes was confirmed, thus eliminating these tissues as useful sources for further investigation of mutant sarcosinemic phenotypes and genotypes. The response in plasma of sarcosine and glycine, after sarcosine loading, distinguished the normal subject from the subjects who were presumably homozygous and heterozygous for the hypersarcosinemia allele. Sarcosine clearance from plasma was delayed greatly (t(1/2), 6.1 hr) in the presumed homozygote and slightly (t(1/2), 2.2 hr) in the presumed heterozygote, while plasma glycine remained constant in the former and rose in the latter. Normal subjects clear sarcosine from plasma rapidly (t(1/2), 1.6 hr) while their plasma glycine trend is downward. The phenotypic responses suggest that hypersarcosinemia is an autosomal recessive trait in this pedigree. Renal tubular transport of sarcosine was normal in the proband even though he presumably lacked the sarcosine oxidation which should normally occur in kidney. Sarcosine catabolism is thus not important for its own renal uptake. Sarcosine interacts with proline and glycine during its absorption in vivo. Studies in vitro in rat kidney showed that sarcosine transport is mediated, saturable, and energy dependent. Sarcosine has no apparent transport system of its own; it uses the low K(m) transport systems for L-proline and glycine to a minor extent and a high K(m) system shared by these substances for the major uptake at concentrations encountered in hypersarcosinemia. Intracellular sarcosine at high concentration will exchange with glycine on one of these systems, which may explain a paradoxical improvement in renal transport of glycine after sarcosine loading in the hypersarcosinemic proband.

In vitro Use of Polyethylene-(1,2-14C)-Glycol to Measure Extracellular Space in Renal Cortex Slices from Neonatal, Immature and Adult Mammals

Polyethylene-(1, 2-14C)-glycol is a reliable probe of the extracellular space in renal cortex slices. The PEG distribution volume is similar to that measured with inulin (24–25% of wet slic