Renal cortical interstitium and fluid absorption by peritubular capillaries.

Every minute, the cortical peritubular capillaries in a 1-g rat kidney take up more than 0.5 ml tubular reabsorbate. Studies of renal lymph and measurements of pressure in capillaries (Pc) and interstitium (Pi) indicate that normally the protein colloid osmotic pressure of peritubular capillary plasma (COPp) provides the necessary absorptive force, keeping Pi at 2-4 mmHg, i.e., 8-10 mmHg lower than Pc. At reduced COPp, continued delivery of fluid from the tubules automatically raises Pi to maintain capillary fluid uptake. The transient Pi response to sudden exposure of the kidney to subatmospheric pressure shows that such adjustment of forces may take place in only 5 s. Most remarkable, adjustment of forces may take place in only 5 s. Most remarkable, reabsorption continues during protein-free perfusion of the isolated rat kidney, apparently effected by a Pi exceeding Pc. A relative retardation of interstitial uptake of ferritin from plasma in this case suggests fluid reabsorption through both small and large pores in the capillary wall. Collapse of the capillaries is presumably prevented by tight tethering to the capillary wall, giving the narrow interstitium a very low compliance.

Microfilament disruption occurs very early in ischemic proximal tubule cell injury.

Experimental ischemic acute renal failure results in disruption of proximal tubule apical membranes. Previous work utilizing immunofluorescence with an anti-actin antibody has demonstrated that the apical cytoskeleton of proximal tubule cells is disrupted during ischemic injury. In this study, using rhodamine-phalloidin which stains only filamentous actin, we demonstrate that graded durations of ischemia resulted in progressive disruption of proximal tubule apical microfilaments. Quantification using spectrofluorometry showed that 5, 15 and 50 minutes of ischemia resulted in 32.8 +/- 4%, 48.8 +/- 2.5%, and 58.4 +/- 2.6% decreases in apical F-actin relative to controls. Ischemia did not qualitatively affect either glomerular or distal tubule F-actin structure, though there were nonprogressive increases in glomerular fluorescence. In summary, rhodamine-phalloidin staining can be used to qualitatively and quantitatively assess proximal tubule microfilaments in vivo. We conclude that ischemia results in very early loss of proximal tubule apical microfilaments, with the majority of F-actin loss occurring within five minutes.

An enzyme immunoassay screening test for the detection of total antinuclear antibodies.

The enzyme immunoassay antinuclear antibody (EIA ANA) screening test method is a new assay format for the qualitative determination of antinuclear antibodies (ANAs) in human serum or plasma. This assay method collectively detects ANAs against double stranded DNA (dsDNA), SS-A/Ro, SS-B/La, Scl-70, Sm, and Sm/RNP antigens, along with serum positive for peripheral, homogeneous, speckled, nucleolar, and centromere patterns. This assay correlated well with data obtained with hemagglutination tests for specific ANA antigens, with the indirect immunofluorescence (IFA) ANA HEp-2 test, and with the Crithidia luciliae IFA test for anti-dsDNA. This new test procedure is both highly specific and sensitive and substantially decreases the time involved when screening large numbers of patient samples.

Effect of acute metabolic acidosis on ammonia metabolism in kidney.

To understand the mechanisms that initiate the increase in ammonia formation during acute acidosis in kidney [amino-15N]- and [amino-15N]glutamine were used as substrates in isolated perfused rat kidney experiments. Perfused kidneys from methionine sulfoximine-treated rats take up glutamine nitrogen at the rate of 1.50 +/- 0.08 mumol.g kidney-1.min-1 while forming ammonia at a rate of 0.65 +/- 0.09 mumol.g.kidney-1.min-1. Mass spectrometer analysis of the perfusate and urine reveals that ammonia is formed from the amide nitrogen of glutamine at the rate of 0.32 +/- 0.06 mumol.g kidney-1.min-1 and ammonia is formed from glutamate derived from glutamine at the rate of 0.21 +/- 0.04 mumol.g kidney-1.min-1. The balance of the ammonia formed is from unidentified endogenous sources. Addition of HCl to the perfusate to lower perfusate pH increases ammonia formation to 1.09 +/- 0.10 mumol.g kidney-1.min-1. The results exclude a role for the purine nucleotide cycle during acute acidosis and confirm that ammonia formation from glutamate derived from glutamine is via glutamate dehydrogenase. Lowering perfusate pH increases the rate of glutamine deamidation significantly by 0.33 +/- 0.06 mumol.g kidney-1.min-1 and increases the rate of ammonia formation via glutamate dehydrogenase insignificantly by only 0.08 +/- 0.04 mumol.g kidney-1.min-1, whereas ammonia formation from endogenous sources remains unchanged. The results demonstrate that regulation of glutamine deamidation is an important controlling step in ammonia formation during acute metabolic acidosis in kidney.

NMR monitoring of phosphate metabolism of rat skeletal muscle during hemorrhage and resuscitation.

Phosphorus nuclear magnetic resonance (NMR) spectroscopy allows noninvasive monitoring of intracellular high-energy metabolites. In the present study we used topical NMR to monitor intracellular levels of ATP, creatine phosphate (CrP), inorganic phosphate (Pi), and pH in the biceps femoris muscle of rats during hemorrhagic shock and resuscitation. Twelve rats weighing 300-500 gm were anesthetized and bled to a mean arterial pressure (MAP) of 50-55 mm Hg for 90 minutes. Then they were resuscitated with lactated Ringers' until MAP returned to normal or resuscitation fluid equaled four times the shed blood volume. During resuscitation, the rats fell into one of two groups: survivor group (n = 5) which could be successfully resuscitated for 60 minutes or longer; or nonsurvivor group (n = 7) which died during resuscitation. In both groups, ATP levels were maintained during hemorrhage and resuscitation. Intramuscular pH dropped about 0.2 pH units in both groups at the end of hemorrhage; however, pH was restored back toward baseline in the survivor group. CrP levels were lower in the nonsurvivor group at the end of hemorrhage. After resuscitation, CrP returned to nearly baseline levels in the survivor group; in the nonsurvivor group, CrP was further depleted after resuscitation. Pi levels were increased in both groups at the end of hemorrhage, but in the survivor group Pi decreased during the first 15 minutes of resuscitation; in the nonsurvivor group Pi increased further to four times baseline levels. This study demonstrated that topical NMR can quantitate a metabolic deficit in skeletal muscle during hemorrhage and resuscitation. The results show that improvement of intracellular Pi and CrP levels correlated with survival.

McArdle's disease heterozygotes. Metabolic adaptation assessed using 31P-nuclear magnetic resonance.

Two daughters of a propositus with documented McArdle's disease were shown by enzyme assay, gel electrophoresis, and immunoblotting to be partially deficient in skeletal muscle phosphorylase and, presumably, heterozygous for the trait. Both exhibited only the adult form of the skeletal muscle isozyme. By 31P-nuclear magnetic resonance, both heterozygotes showed a greater production of acid during fully aerobic exercise than when blood flow was occluded in ischemic exercise. This pattern is in contrast to that of control subjects, where there is significantly greater acid production in ischemic versus aerobic exercise, and distinct from that of phosphorylase-negative patients in which no acid is produced in either circumstance. We suggest that these heterozygotes may have adapted to their diminished phosphorylase by enhancing utilization of plasma glucose. If so, this mechanism could account for the observation that most of the symptoms of McArdle's disease are often manifest only in adulthood. These studies also show that although there are very high concentrations of phosphorylase in skeletal muscle (approximately 2% of the soluble protein), such a high level is essential for normal muscle glycogenolysis.

Localization of phosphorus metabolites and sodium ions in the rat kidney.

Relative amounts of phosphorus-containing metabolites and sodium ions present in different regions of the in vivo rat kidney were obtained using a surface-coil probe and recently developed NMR rotating-frame methods. During altered physiologic states, changes in distribution of metabolites and sodium ions within the kidney were identified in one-dimensional metabolite maps. This technique may have important applications to disorders commonly found in clinical medicine.

Diabetic nephropathy. Is end-stage renal disease inevitable?

The appearance of proteinuria in an insulin-dependent diabetic patient is an ominous sign. Proteinuria heralds the presence of diabetic nephropathy and early death, or chronic renal failure requiring dialysis or transplantation, in 50% of patients. The pathogenesis of diabetic nephropathy is unknown. Adequate insulin administration is the most important preventive measure. Hypertension, if present, should be aggressively treated to delay progression of renal disease. Good nutrition, prompt treatment of urinary tract infections, and caution in the use of radiocontrast agents are other important preventive measures. Hemodialysis, peritoneal dialysis, and transplantation are options for patients with end-stage renal disease. No matter which is selected, the patient may still have multiple amputations, blindness, congestive heart failure, infections, and uncontrolled glycemia. Advancements are being made, however, that promise a better future for insulin-dependent diabetics.

The relationship between glutamate deamination and gluconeogenesis in kidney.

The effect of 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32], was tested on NH3 formation via the purine nucleotide cycle and glutamate dehydrogenase (EC 1.4.1.2). NH3 excretion in rats increased 70-fold after 48 h of NH4Cl feeding, from 12.2 +/- 4.5 to 862 +/- 190 mumol/mg of creatinine. At 4 h after a single intraperitoneal injection of 3-mercaptopicolinate into NH4Cl-fed rats, NH3 excretion was inhibited by 93%. Kidneys of NH4Cl-fed plus 3-mercaptopicolinate-treated rats, compared with those of NH4Cl-fed rats, showed a 3.5-fold increase in the content of IMP, 5-fold increase in adenylosuccinate, 4-fold increase in aspartate, and a 30% increase in AMP. 3-Mercaptopicolinate completely inhibited NH3 and glucose formation from glutamate in tubules from acidotic rats and NH3 formation from aspartate in kidney perfusion experiments. When transamination in tubules was prevented by 2-amino-4-methoxy-trans-but-3-enoic acid, formation of glucose, but not of NH3, from glutamate was inhibited. 3-Mercaptopicolinate completely inhibited NH3 formation from aspartate in the presence of the aminotransferase inhibitor in kidney tubules. The data show that NH3 can be formed via glutamate dehydrogenase and the purine nucleotide cycle at significant and approximately equal rates. 3-Mercaptopicolinate has no direct effect on NH3 formation via glutamate dehydrogenase, but inhibits that via the purine nucleotide cycle. We conclude that gluconeogenesis is not regulatory for NH3 formation in kidney.

Early events in the initiation of ammonia formation in kidney.

Experiments were designed to examine the early events in the initiation of glutamate deamination in kidney. Perfused kidneys from methionine sulfoximine-treated rats formed ammonia from [15N]glutamate via the purine nucleotide cycle. The turnover of the 6-amino group of adenine nucleotides to yield ammonia occurred at the rate of 0.30 mumol/g of kidney/min. This rate is 3-4 times larger than in liver and is in agreement with published rates of the purine nucleotide cycle in kidney. The addition of 0.1 mM fluorocitrate to glutamate perfusions stimulated ammonia formation 3 1/2-fold. The turnover of the 6-amino group of adenine nucleotides increased during the first 5 min after adding fluorocitrate to form ammonia predominately from tissue glutamate and aspartate. This turnover correlates with a 3 1/2-fold increase in kidney tissue IMP levels. As the ATP/ADP ratio fell the purine nucleotide cycle was inhibited and glutamate dehydrogenase was stimulated to form ammonia stoichiometric with glutamate taken up from the perfusate. Ammonia formation via glutamate dehydrogenase occurred at a rate of 1.0 mumol/g of kidney/min. Fluorocitrate completely blocked ammonia formation from aspartate in perfusions. The perfused kidney formed ammonia from aspartate via the purine nucleotide cycle at a rate of 1.0 mumol/g of kidney/min. The results indicate a discrete role for aspartate in renal metabolism. Ammonia formation via the purine nucleotide cycle can occur at significant rates and equal to the rate of ammonia formation from glutamate via glutamate dehydrogenase.

The purine nucleotide cycle in the regulation of ammoniagenesis during induction and cessation of chronic acidosis in the rat kidney.

The effect of chronic acid feeding and its subsequent withdrawal was determined on the amounts of the metabolic intermediates and enzymic activities of the purine nucleotide cycle. Sprague-Dawley rats were given 1.5% (w/v) NH4Cl in their drinking water for 5 days. The renal excretion of NH3 rose 70-fold and the rats developed acidosis. The amount of renal IMP rose from a control value of 4.5 +/- 2.2 to 20.4 +/- 3.7nmol/g of kidney after 48h of acid feeding (P less than 0.001) and fell to normal within 48h of the recovery. Adenylosuccinate concentrations fell from a control value of 4.5 +/- 0.9nmol/g of kidney to 1.2 +/- 0.3nmol/g (P less than 0.005) by day 5 of acidosis and continued to fall to undetectable values by 48h after recovery. The amount of AMP remained constant through the acid-feeding and the recovery periods. The activity of adenylosuccinate synthetase, the rate-limiting enzyme of the purine nucleotide cycle, paralleled the rise and fall in NH3 excretion. The activities of phosphate-dependent glutaminase and glutamate dehydrogenase were elevated during the acid-feeding and the recovery period. Thus changes in the purine nucleotide cycle correlate with changes in NH3 excretion to a more parallel degree than does the activity of glutaminase or glutamate dehydrogenase.

The purine nucleotide cycle. A pathway for ammonia production in the rat kidney.

Particle-free extracts prepared from kidney cortex of rat catalyze the formation of ammonia via the purine nucleotide cycle. The cycle generates ammonia and fumarate from aspartate, using catalytic amounts of inosine monophosphate, adenylosuccinate, and adenosine monophosphate. The specific activities of the enzymes of the cycle are 1.27+/-0.27 nmol/mg protein per min (SE) for adenoylosuccinate synthetase, 1.38+/-0.16 for adenylosuccinase, and 44.0+/-3.3 for AMP deaminase. Compared with controls, extracts prepared from kidneys of rats fed ammonium chloride for 2 days show a 60% increase in adenylosuccinate synthetase and a threefold increase in adenylosuccinase activity, and a greater and more rapid synthesis of ammonia and adenine nucleotide from aspartate and inosine monophosphate. Extracts prepared from kidneys of rats fed a potassium-deficient diet show a twofold increase in adenylosuccinate synthetase and a threefold increase in adenylosuccinase activity. In such extracts the rate of synthesis of ammonia and adenine nucleotide from aspartate and inosine monophosphate is also increased. These results show that the reactions of the purine nucleotide cycle are present and can operate in extracts of kidney cortex. The operational capacity of the cycle is accelerated by ammonium chloride feeding and potassium depletion, conditions known to increase renal ammonia excretion. Extracts of kidney cortex convert inosine monophosphate to uric acid. This is prevented by addition of allopurinol of 1-pyrophosphoryl ribose 5-phosphate to the reaction mixture.