Myong Won Kahng

Changes in mitochondrial lipids of rat kidney during ischemia

Lipid changes in mitochondria isolated from rat kidney after various periods of ischemia were analysed by thin-layer chromatography and gas-liquid chromatography. Free fatty acids were increased at 30 min and more so thereafter. Total phospholipid fatty acids decreased steadily. The proportion of diphosphatidylglycerol (cardiolipin) in the total phospholipid fraction decreased at 30 min, but the proportion of phosphatidylcholine and phosphatidylethanolamine in the total phospholipid fraction did not change until the irreversible phase of ischemic injury. We have shown that decrease of cardiolipin in mitochondrial membrane occurs early during ischemia, and only during the irreversible phase of ischemia are phosphatidylethanolamine and phosphatidylcholine broken down. It is postulated that these phenomena are due to activation of phospholipase in the mitochondrial membrane.

High-affinity renal lead -binding proteins in environmentally-exposed humans

Chronic low level lead (Pb) exposure is associated with decrements in renal function in humans, but the molecular mechanisms underlying toxicity are not understood. We investigated cytosolic Pb-binding proteins (PbBP) in kidney of environmentally-exposed humans to identify molecular targets of Pb and elucidate mechanisms of toxicity. This study is unique in that it localized PbBPs based on physiologic Pb that was bound in vivo. Two Pb-binding polypeptides were identified, thymosin beta 4 (T beta 4, 5 kDa) and acyl-CoA binding protein (ACBP, 9 kDa, also known as diazepam binding inhibitor, DBI). These polypeptides, which have not been previously recognized for their metal-binding capabilities, were shown to bind Pb with high affinity (Kd approximately 14 nM) and to account for an estimated > 35% of the total Pb in kidney cortex tissue. Both T beta 4 and ACBP (DBI) occur across animal species from invertebrates to mammals and in all major tissues, serving multiple possible functions (e.g. regulation of actin polymerization, calmodulin-dependent enzyme activity, acyl-CoA metabolism, GABA-A/benzodiazepine receptor modulation, steroidogenesis, etc.). Thus, these data provide the first evidence of specific molecular targets of Pb in kidney of environmentally-exposed humans, and they suggest that low-level Pb toxicity may occur via alteration of T beta 4 and ACBP (DBI) function in renal and other tissues, including the central nervous system.

Metabolic and ultrastructural response of rat kidney cortex to in vitro ischemia

Abstract The time course of changes in metabolite concentrations and ultrastructure during ischemia was studied in rat kidney cortex by incubating tissues at 37°C for 15, 30, 60, and 120 min or at 0°C for 0.5, 6, 12, 24, 48, 72, and 96 hr. Ischemia at 37°C for 15 min caused a rapid fall of ATP (13% of control) and ADP (42%) with a concomitant increase in AMP (2.9-fold) and P 1 (2.8-fold). At 60 and 120 min, AMP also decreased resulting in the decrease of total adenine nucleotide pool. At 0°C, ATP was 50% of control at 0.5 hr and was maintained at this level up to 72 hr. The changes in other adenine nucleotides were also moderate at 0°C. The accumulation of lactate was the highest at all time intervals studied (at 37°C, 11-fold at 15 min and 26-fold at 120 min; at 0°C, 5-fold at 0.5 hr and 14-fold at 96 hr). The two rate limiting enzymes of glycolysis, i.e., phosphofructokinase and pyruvate kinase were activated during ischemia to different extents depending on the temperature and length of time of ischemia based on the metabolite patterns. At 37°C, swelling of mitochondria with loss of matrix granules and the formation of flocculent densities in proximal tubule cells were the prominent ultrastructural features which progressed with time. At 0°C, swelling, cytoplasmic clumping and dilatation of endoplasmic reticulum were observed. Mitochondrial flocculent densities appeared at 96 hr. Based on metabolic and ultrastructural observations, 1 hr of ischemia at 37°C and 72 hr at 0°C were found to be the “point-of-no-return” from ischemic injury in renal cortex.

Neuromuscular toxicity of pyridostigmine bromide in the diaphragm, extensor digitorum longus, and soleus muscles of the rat☆

The neuromuscular junctions from diaphragm, soleus, and extensor digitorum longus (EDL) muscles of male albino rats were assessed for morphological alterations following acute (30-min) and subacute (2-day) exposure to pyridostigmine bromide in Mestinon-equivalent buffer. These muscles were selected to compare the effects of the drug on muscles of different fiber type composition. The diaphragm has approximately equal numbers of type I and type II fibers while the soleus and EDL possess primarily type I and type II fibers, respectively. Pyridostigmine was administered to each acute-exposure animal by a single subcutaneous injection of 0.36 mg/kg pyridostigmine and to each subacute-exposure animal by a subcutaneously implanted osmotic minipump containing 10 mg/ml pyridostigmine. Both treatments resulted in whole blood cholinesterase (ChE) depression of approximately 60-70% as determined by radiometric assay. Control animals received only Mestinon-equivalent buffer. Both acute and subacute exposures resulted in morphological alteration of the neuromuscular junctions (NMJs) of all three muscles, although considerable variation in the extent of damage occurred even within individual NMJs. The most frequently observed presynaptic alterations were mitochondrial damage and partial withdrawal of nerve terminal branches (partial denervation). Post-synaptic changes included occasional rarefaction of mitochondrial matrices and disruption of the myofibrillar organization in small numbers of subjunctional sarcomeres. The data indicate that acute or subacute exposure to pyridostigmine bromide at a whole blood ChE depression of 60-70% results in similar alterations to the NMJs of three muscles with substantially different fiber type compositions. Although the severity of the damage varies from fiber to fiber, the variability appears random and not related to a specific fiber type or dosage regimen.

UDP-Glucuronyltransferase Kinetics for 3-Trifluoromethyl-4-nitrophenol (TFM) in Fish

Abstract Studies were conducted to address glucuronidation of 3-trifluoromethyl-4-nitrophenol (TFM) in sea lampreys Petromyzon marinus, channel catfish Ictalurus punctatus, rainbow trout Oncorhynchus mykiss, and bluegills Lepomis macrochirus. The ability of these species to biotransform TFM was investigated by determining the kinetics of UDP-glucuronyltransferase (UDPGT; also known as glucuronosyltransferase) in vitro from hepatic microsomal preparations. Maximal velocity (V max, nmol/min·mg) for UDPGT activity toward TFM was significantly greater (P rainbow trout (97 μM) > channel catfish (172 μM) > sea lamprey (261 μM). Analysis of V max/K m ratios, a measure of enzyme efficiency (nmol/min-mguM TFM), indicated that the efficiency of UDPGT activities in all species examined was in...

Metabolic studies of postischemic acute renal failure in the rat.

Postischemic acute renal failure was induced by 1 hr of clamping of the renal vasculature. Adenine nucleotide (ATP, ADP, AMP) and lactate (Lac) levels were measured after 0, 0.25, 1, 6, 24, and 48 hr of reflow to determine the time necessary for recovery to control levels. After 1 hr of ischemia with no reflow, [ATP] was 18% and [Lac] was 10-fold control levels. Control levels were restored after 24 hr of reflow. Variable ischemic times (5, 15, 30, 60, 90, and 120 min) followed by (1) no reflow or (2) 24 hr of reflow were also studied. [ATP] decreased to 25 and 13% of controls after 5 and 120 min of ischemia, respectively, and [Lac] increased to 5- and 13-fold controls after 5 and 120 min. Five to ninety minutes of ischemia followed by 24 hr of reflow resulted in a trend toward restoration of ATP and Lac levels; whereas, 120 min of ischemia followed by 24 hr of reflow resulted in death. The results indicate that: (1) In vivo ischemia results in a drastic and rapid shift in the ATP-ADP-AMP equilibrium; (2) the absolute concentration of ATP is not a reliable criterion of cell viability, but the ability to resynthesize ATP may be determinant in the reversibility of the lesion; (3) 1 hr of ischemia is reversible with respect to restoration of [ATP] and [Lac], but 24 hr of reflow are needed for restoration; and (4) ischemia for 90 min results in a metabolic derangement which is partially reversible in that metabolite levels are partially restored after 24 hr of reflow. However, 90 min of vascular clamping is not functionally reversible since the majority of animals exhibit severe azotemia and do not survive.

Metabolic studies of HgCl2-induced acute renal failure in the rat

HgCl2 was used to produce acute renal failure (ARF) in rats. It caused significant decreases (P < 0.001) in ATP, ADP, and total adenine nucleotide levels to 50% of controls at 48 hr with no change in AMP levels. Lactate increased to threefold control levels at 6 hr and remained significantly elevated (P < 0.001) at 48 hr. At 24 hr, there was widespread necrosis in the pars convoluta and pars recta. At 48 hr, necrosis was accompanied by some regeneration, and creatinine levels exceeded 5 mg/dl. Dithiothreitol (30.8 mg/kg, ip, 30 min after HgCl2) partially ameliorated the functional lesion (creatinine levels ⩽3.6 mg/dl) and resulted in reduced necrosis in the pars convoluta, but did not significantly alter the metabolic pattern. The results indicate an early metabolic disturbance and are consistent with the idea that HgCl2 exerts a direct action on the enzymes of the mitochondrial electron transport chain. The failure of dithiothreitol to alter the metabolic pattern of HgCl2-induced ARF indicated that its protective effect was probably not mediated directly through the maintenance of adenine nucleotide levels.

Metabolic studies of glycerol-induced acute renal failure in the rat

Abstract Glycerol was used to induce acute renal failure (ARF) in rats. It caused a decrease of kidney ATP levels to 48% of control at 6 hr which remained low up to 48 hr. ATP + ADP + AMP(AXP) was 55% of control at 6 and 24 hr and 64% at 48 hr. Lactate levels were fourfold control levels at 0.25, 1, and 6 hr, and twofold at 24 and 48 hr. Hemoglobinemia and hemoglobinuria occurred within 0.25 hr. By light microscopy, casts at 6 hr, necrosis of renal cortex at 24 hr, and necrosis with some regeneration at 48 hr were seen. Fructose at the same dose also lowered ATP and AXP levels to a similar degree as glycerol within the same time intervals, but hemoglobinemia, hemoglobinuria, necrosis, or ARF was not evident. Dihydroxycetone at the same dose maintained ATP and AXP at control values with no hemoglobinemia, hemoglobinuria, necrosis, or ARF. Both fructose and dihydroxyacetone resulted in high lactate levels (sevenfold) within 0.25 hr. Saline loading prior to glycerol infection ameliorated ARF but did not improve the AXP levels or the morphologic lesion. Results indicate that glycerol-induced ARF is not solely the result of a hyperosmolar effect, adenosine release, casts, or pigments. The changes in AXP (particularly ATP) levels reflect an early metabolic disturbance which is part of the process and may predispose toward ARF in the presence of additional factors.

Characterization of an in vitro system of human renal papillary collecting duct cells

SummaryWe have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 104 live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOsm for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and γ-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins.

Substrate Oxidation and Enzyme Activities of Ketone Body Metabolism in the Developing Pig

Utilization of β-hydroxybutyrate-3-14C was studied in developing pig brain, heart and kidney slices by following the incorporation of 14C into CO2. All three tissues s