Proteomic analysis of rat kidney cortex following treatment with gentamicin

The regionally specific structure and function of the kidney renders it susceptible to toxic exposure. To characterize these changes at the proteome level, we have investigated the effects on protein expression following treatment with gentamicin. The more than 20 proteins identified were involved in the citric acid cycle, gluconeogenesis, fatty acid synthesis, and transport or cellular stress responses. These results strongly support the notion that energy production is impaired and mitochondrial dysfunction is involved in gentamicin-induced nephrotoxicity.     

Copper accumulation in the soluble and particulate fractions of renal cortex in the streptozotocin-diabetic rat

The accumulation and subcellular distribution of copper in the kidney of streptozotocin-diabetic rats were investigated. Male Sprague-Dawley rats received streptozotocin (50 mg/kg body wt on two consecutive days) intraperitoneally and were fed either commercial or purified diet. The concentrations of copper, zinc, iron, and manganese present in intact kidney, renal cortex, and renal medulla were compared at various times. Chow-fed diabetic rats had a renal copper concentration 2.6 times greater than age-matched controls after 2 weeks. The concentration of zinc was only 30% higher in diabetic kidney than in control tissue, whereas the iron and manganese concentrations were similar for both groups. The additional complement of renal copper was localized entirely in the cortex and was significantly reduced by oral treatment with penicillamine, a copper chelating agent. When diabetic rats were fed purified diet (15-20 ppm Cu), the quantity of copper accumulated in the renal cortex increased from 2.3 to 8.7-fold higher than in control tissue from 1 to 4 weeks, respectively, after injection with streptozotocin. Copper levels in. both the soluble and particulate (165, 000g pellet) fractions of diabetic renal cortex were similarly increased at each time. Gel filtration Chromatographic analysis of the cytosol showed that all of the copper accumulated in the soluble fraction was associated with metallothionein. The distribution of excess copper in the particulate fraction was determined by differential centrifugation. The additional quantity of metal was localized in the crude nuclear fraction of renal cortex in the diabetic rat. Further analysis revealed that the lysosomal fraction from 3-weeek diabetic rats had a copper level 16-fold higher than in the controls. The possibility that accumulation of excessive levels of copper in the streptozotocin-diabetic kidney may contribute to the development of diabetic nephropathy is discussed.     

Uptake of mercury and mercury-amino acid complexes by rat renal cortex slices.

This study was undertaken in order to assess the effects of metabolism and complexations with amino acids on the renal uptake of mercury using rat renal cortex slices as the experimental system. Mercury levels attained in the slices after 60 min of incubation were 50% higher with mercuric cysteine than with mercuric chloride. This enhancement of uptake with mercuric cysteine was reduced in the presence of a tenfold molar excess of histidine or lysine, but not by serine. Excess cysteine markedly increased mercury uptake. Incubation at 25 degrees significantly reduced uptake of mercuric cysteine, but not mercuric chloride. Anaerobic conditions and incubation in the presence of DNP each reduced mercuric cysteine uptake to the control level of mercuric chloride without affecting uptake of mercuric chloride. The differential aspects of metabolism on the uptake of mercuric cysteine and mercuric chloride and the competitive effects obtained with amino acids known to compete with cysteine in renal reabsorption support the hypothesis that a portion of the renal uptake of mercury operates through amino acid transport mechanisms acting on mercury-amino acid complexes.     

Lithium inhibits muscarinic-receptor-stimulated inositol tetrakisphosphate accumulation in rat cerebral cortex.

The effects of Li+ on carbachol-stimulated phosphoinositide metabolism were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. The muscarinic agonist carbachol evoked an enhanced steady-state accumulation of [3H]inositol monophosphate ([3H]InsP1), [3H]inositol bisphosphate ([3H]InsP2), [3H]inositol 1,3,4-trisphosphate ([3H]Ins(1,3,4)P3), [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) and [3H]inositol tetrakisphosphate ([3H]InsP4). Li+ (5 mM), after a 10 min lag, severely attenuated carbachol-stimulated [3H]InsP4 accumulation while simultaneously potentiating accumulation of both [3H]InsP1 and [3H]InsP2 and, at least initially, of [3H]Ins(1,3,4)P3. These data are consistent with inhibition of inositol mono-, bis- and 1,3,4-tris-phosphate phosphatases to different degrees by Li+ in brain, but are not considered to be completely accounted for in this way. Potential direct and indirect mechanisms of the inhibitory action of Li+ on [3H]InsP4 accumulation are considered. The present results stress the complex action of Li+ on cerebral inositol metabolism and indicate that more complex mechanisms than are yet evident may regulate this process.     

Properties of γ-aminobutyric acid synthesis by rat renal cortex

Substantial synthesis of γ-aminobutyric acid occurs in rat renal cortex. Renal glutamate decarboxylase activity (24.3±2.9 (S.E.) nmols/mg protein per h) is 15% of that in brain; renal γ-aminobutyric acid content (39.5±5.3 (S.E.) nmols/g wet wt.) is 5% of the whole brain concentration. Properties of glutamate decarboxylase were studied in homogenates of rat renal cortex and rat brain under conditions for which γ-aminobutyric acid formation from [2,3-³H]glutamate and CO₂ release from [1-¹⁴C]glutamate were equal. Several properties of renal glutamate decarboxylase distinguish it from the corresponding brain enzyme: (1) renal glutamate decarboxylase is selectively inhibited by cysteine sulfinic acid (K_i = 5·10^?5 M) ; (20 renal glutamate decarboxylase is less sensitive (K_i = 3–5·10^?5 M)_to inhibition by aminooxyacetic acid than is the brain enzyme (K_i = 1·10^?6 M); (3) brain but not renal glutamate decarboxylase activity can be substantially stimulated in vitro by the addition of exogenous pyridoxal 5′-phosphate; (4) renal glutamate decarboxylase is significantly decreased in renal cortex from rats on a low-salt diet. Proximal tubules are enriched in glutamate decarboxylase compared to the activity in whole renal cortex or glomeruli (42, 22 and 14 nmols/mg protein per h, respectively). We speculate that renal γ-aminobutyric acid synthesis does not reflect the presence of GABAergic renal nerves, but may serve a function in proximal tubular cells.     

Purification of brush border membrane vesicles from rat renal cortex by size-exclusion chromatography.

Size-exclusion chromatography with controlled pore glass (CPG) was used in the further purification of renal brush border membrane vesicles (BBMV) isolated by the Ca precipitation method. The BBMV obtained had an almost spherical shape and their average diameter was about 95 nm in isotonic solution. The specific activities of alkaline phosphate and leucine aminopeptidase in the BBMV preparation were increased 18- and 17-fold, respectively, over those in the crude homogenate. The uptake of D-glucose by the purified BBMV in the presence of a sodium gradient reached 8.53 nmol/mg protein at 20 s. These results indicate that CPG chromatography is suitable procedure by which to obtain purified renal BBMV of homogenous size and with high specific marker enzyme activity for use in the study of membrane transport.     

The effect of diamide on amino acid transport by rat renal cortex slices.

Diamide directly added to renal cortical slices inhibits the uptake of amino acids. Steady-state kinetic analysis indicates an inhibition of alpha-amino acid influx without effect on efflux. The effect could be reversed by addition of pyruvate to the incubation medium. Although there was a good correlation of the transport effect of diamide with its ability to decrease cellular reduced glutathione concentration, there did not appear to be a necessary connection between them. This was shown by the fact that renal cortical slices stored at 4 degrees C have no alteration in amino acid uptake despite the fact that GSH concentration is as low as that seen with diamide. Diamide was shown to have a direct effect on the uptake of glycine by isolated renal brush border membrane vesicles.     

On the development of glycine transport systems by rat renal cortex

The initial uptake of glycine by renal cortical slices from newborn Sprague-Dawley and Long-Evans rats is the same as that observed in adult tissues. Both newborn and adult tissue possess similar high and low affinity glycine transport systems which require an examination of velocity measurements over a wide range of concentration (0.02--50.0 mM) for their discernment. Initial rates of glycine uptake by isolated renal tubule fragments from newborn and adults are similar at a physiological substrate concentration but at high glycine levels there appears to be a decrease in velocity of uptake (V) associated with the high Km system in the young. Whatever preparation of renal cortex is studied, there is a consistent finding that immature tissue is able to accumulate much higher intracellular levels of glycine than the adult, a finding consistent with slower efflux from the cell. An interpretation of the etiology of physiologic aminoaciduria in young animals should take this into account.     

Stimulation of mitochondrial pyruvate transport in rat renal cortex by phenylephrine

Phenylephrine effect on liver and kidney cortex mitochondrial pyruvate concentration was investigated. While in liver the alpha 1-adrenergic agent produced a decrease in pyruvate content, a significant increase was observed in kidney, even in the presence of 0.5 mM alpha-cyano-4-hydroxy-cinnamate. These changes were not observed when pyruvate was formed by intramitochondrial transamination of alanine, suggesting a role for the pyruvate transport across mitochondrial membranes in the regulation of mitochondrial pyruvate metabolism in kidney cortex. This was corroborated measuring the phenylephrine effect on pyruvate carboxylation.     

Oxalate accumulation in rat renal cortical slices

Tubular transport of oxalate is thought to be an energy-mediated process which may contribute to the renal deposition of calcium oxalate in a variety of pathologic states. In order to examine this possibility, the renal handling of oxalate was investigated in rat renal cortical slices in vitro. Slices incubated in vitro with 1 microM [14C]oxalate in Krebs-Ringer bicarbonate buffer at 25 degrees C for 180 min achieved a mean slice to medium ratio of 2.8 +/- 0.08 (SEM) and a mean tissue concentration of 7.7 +/- 0.2 mumol/kg dry wt (N = 64). Section freeze-dry autoradiographs demonstrated maximum uptake within proximal tubule cells but no crystals were evident. Substituting N2 for O2, adding KCN, or removing Ca2+ increased uptake of 14C-oxalate. Dinitrophenol (DNP) and iodoacetamide (IoAc), however, significantly decreased, and O degrees C eliminated slice uptake. Slices incubated with 100 microM [14C]oxalate showed a further increase in tissue accumulation and the appearance of [14C]oxalate crystals. Crystals formed in vitro were deposited throughout the tissue. Oxalic acid did not appear to share the organic acid by renal cortical slices in vitro is largely independent of energy-mediated mechanisms.     

Oxygen consumptions and potassium contents of slices of rat renal cortex.

An oxygen electrode respirometer for determining the oxygen consumption of slices of mammalian renal cortex is described and assessed. Though rat renal cortical slices incubated in potassium-free medium for one hour lost 102 +/- 14 mmol of potassium/kg dry weight, there was only a small, nonsignificant fall in oxygen consumption. In contrast the oxygen consumption of slices incubated in potassium-free medium with 10 mmol.1-1 ouabain was markedly reduced (by 32 +/- 6%), while such slices lost 180 +/- 15 mmol of potassium/kg dry weight. These disproportionate effects on potassium loss and inhibition of oxygen consumption suggest that in renal cortical slices the loss of potassium in low potassium medium is not primarily due to inhibition of the conventional sodium pump.     

A study of calcium pump activity of lysosomes from rat renal cortex.

Fractions rich in either primary or secondary lysosomes were prepared from rat renal cortex by differential centrifugation and evaluated for their capacity for net calcium uptake. No uptake was observed in the absence of ATP. A vigorous uptake did take place in the presence of ATP but it was largely prevented by azide and other inhibitors of mitochondrial calcium uptake, suggesting that it was attributable to contamination by mitochondria. Evidence was obtained for an inhibitory influence of the secondary lysosomal fraction on mitochondrial calcium uptake.     

Specificity determinants of rat tissue kallikrein probed by site-directed mutagenesis.

Site-specific mutagenesis was employed to study structure-function relationships at the substrate binding site of rat tissue kallikrein. Four kallikrein mutants, the Pro219 deletion (P219del), the 34-38 loop Tyr-Tyr-Phe-Gly to Ile-Asn mutation [YYFG(34-38)IN], the Trp215----Gly exchange (W215G) and the double mutant with Tyr99----His and Trp215----Gly exchange (Y99H:W215G) were created by site-directed mutagenesis to probe their function in substrate binding. The mutant proteins were expressed in Escherichia coli at high levels and analyzed by Western blot. These mutant enzymes were purified to apparent homogeneity. Each migrated as a single band on SDS-PAGE, with slightly lower molecular mass (36 kDa) than that of the native enzyme, (38 kDa) because of their lack of glycosylation. The recombinant kallikreins are immunologically identical to the native enzyme, displaying parallelism with the native enzyme in a direct radioimmunoassay for rat tissue kallikrein. Kinetic analyses of Km and kcat using fluorogenic peptide substrates support the hypothesis that the Tyr99-Trp215 interaction is a major determinant for hydrophobic P2 specificity. The results suggest an important role for the 34-38 loop in hydrophobic P3 affinity and further show that Pro219 is essential to substrate binding and efficient catalysis of tissue kallikrein.     

Regulatory characteristics of amino acid transport in newborn rat renal cortex cells.

The uptaken of alpha-aminoisobutyric acid by slices of kidney cortex from newborn rats is enhanced by a preliminary incubation of the tissue in buffer at 37 degrees C. This effect is abolished by anaerobiosis, the presence of dinitrophenol or the removal of Na+ during the preliminary incubation. Cycloheximide (50 muM) and purimycin (1 mM) as well as alpha-aminoisobutyric acid, glycine and proline (5 mM) in the preincubation buffer also abolish the effect, while actinomycin D (0.8 muM) partially the phenomenon indicates that the enhanced uptake is due to an increased entry rate into the cells without a change in effux. There is no alteration in the apparent transport Km but an increase in the V for entry. The effect is dependent on tissue age being observed between birth and 22 days, after which there is a decrease in response to preliminary incubation with no effect seen in adult tissues.     

The effect of diamide on amino acid transport by rat renal cortex slices

Diamide directly added to renal cortical slices inhibits the uptake of amino acids. Steady-state kinetic analysis indicates an inhibition of α-amino acid influx without effect on efflux. The effect could be reversed by addition of pyruvate to the incubation medium. Although there was a good correlation of the transport effect of diamide with its ability to decrease cellular reduced glutathione concentration, there did not appear to be a necessary connection between them. This was shown by the fact that renal cortical slices stored at 4°C have no alteration in amino acid uptake despite the fact that GSH concentration is as low as that seen with diamide. Diamide was shown to have a direct effect on the uptake of glycine by isolated renal brush border membrane vesicles.     

Ultrastructure of distal nephron cells in rat renal cortex

Distal nephron segments in the rat renal cortex contain distal convoluted tubule cells (DCT cells), connecting tubule cells (CNT cells), intercalated cells (I cells), and principal cells (P cells). The present study was carried out to expand present knowledge on the ultrastructure of these cells. The cells were sampled from superficial cortex and analyzed by electron microscopy. Several morphometric parameters were determined and statistical comparison between cell types was performed. Significant structural differences between the cell types were demonstrated. DCT cells showed the highest volume density of mitochondria whereas the amplification of basolateral membranes was higher in CNT cells than in I and P cells. The surface density of the membrane that bounds intermediate vesicles in the apical cytoplasm was twofold higher in I cells than in the other cell types. The morphological differentiation found in the present study adds to available evidence indicating a functional differentiation between the cell types and provides a reference for structure-function correlations in these cells.