This investigation studies the toxicity of heme proteins and/or their break-down products on renal function. Heme proteinemia precedes acute tubule necrosis at a frequency great enough to suggest a causal relationship between the two events. Physiological and metabolic functions of kidney slices are investigated in several models of acute tubule necrosis. Organic acid and organic base transport is depressed earliest. These alterations in tubule function cannot be explained by ischemia or obstruction alone. Heme proteinemia in rats or incubation of renal slices in medium containing heme proteins yields several interesting observations. Neither in vivo or in vitro do hemoglobin and methemoglobin alone produce a depressive effect on the transport systems studied. However, parallel to many clinical situations, when such secondary insults as hypoxia and elevated ammonia concentrations are included in the experimental design, transport functions are depressed. Ferrihemate, a molecule smaller than hemoglobin or methemoglobin, depresses transport function without secondary insults. From these studies it is concluded that heme proteins play a role in tubule dysfunction seen in acute tubule necrosis. A model is presented that collates these data with other factors known to play a part in the pathogenesis of this renal syndrome.

1. While azotaemic sera depressed the in-vitro Na-iodohippurate transport of rat kidney slices at any concentration, normal sera excited transport at low concentrations and depressed transport at high concentrations. The depression of transport by azotaemic sera was partially overcome by neomycin feeding in contrast to the depression by normal sera, which was not altered by neomycin feeding.2. Plotting the reciprocal of sodium-iodohippurate accumulated by slices against the reciprocal of the media concentrations of sodium iodohippurate suggested that the depression of hippurate transport produced by normal and azotaemic sera was competitive in nature.3. Normal sera slowed the efflux of Na-iodohippurate from kidney slices while azotaemic sera affected it very little.4. The depression produced by normal and azotaemic sera and the stimulation produced by low concentrations of normal sera were seen with serum ultrafiltrates and dialysates, and after passage through cation exchange columns, but not anion exchange columns.5. The effects on Na-iodohippurate accumulation by normal and azotaemic sera could be reproduced with metabolizable (lactate), and nonmetabolizable (hippurate) organic anions as well as combinations of these.6. The implications of these observations on the altered renal transport of Naiodohippurate produced by azotaemic and normal sera are discussed.Azotaemic sera contain dialysable substances that depress organic anion transport as represented by hippurate or para aminohippurate accumulation (Preuss, Massry, Maher, Gilliece

1. The effects were investigated of non-dialysable substances obtained from urines of patients with nephrotic syndrome and those with myelomatosis (heavily laden with light chains) on renal function in a system in vitro. 2. The ability of the rat kidney slice to accumulate hippurate and tetraethylammonium (TEA), and to produce ammonia and glucose, was measured after incubation in urine proteins from ten patients with myelomatosis. All slice functions measured at protein concentrations of 10 mg/ml decreased significantly compared with control. Hippurate accumulation averaged 58%, TEA accumulation, 53%, ammoniagenesis, 59%, and gluconeogenesis, 57% of control. An inverse relationship between protein concentration and hippurate accumulation was noted. 3. Slices incubated in proteins from eight nephrotic patients showed no consistent decrease from control in hippurate accumulation (95%), TEA accumulation (103%), ammoniagenesis (91%) or gluconeogenesis (92%). 4. Since urinary proteins from patients with myelomatosis, unlike urinary proteins from the nephrotic patients, had a consistently deleterious effect on the function of renal slices, this suggests that proteins found in urines from myelomatous patients may play a role in the disturbance of proximal tubular function sometimes seen in this disorder.

Following acute or chronic acid challenge, kidneys produce more ammonia from glutamine ( 1 ) . Increased production of this urinary buffer is the kidneys' chief means for excretion of the excess acid ( 2 ) . Since the mechanisms involved in increasing ammoniagenesis following acidosis are presently unknown, it is important to examine biochemical means for stimulating renal ammoniagenesis.Previous investigators have related ammoniagenesis to citric acid cycle activity in many ways (3)(4)(5). Recently, we reported that inhibitors of citric acid cycle activity could increase ammonia formation from glutamate by isolated dog tubules (6). To gain further knowledge concerning this phenomenon, a series of in vitro studies with rat kidney slices and in vivo studies with rats were performed. These studies show that interruption of the citric acid cycle with either fluorocitrate or malonate stimulates ammoniagenesis in vitro and that injection of these same inhibitors into rats results in increased ammonia excretion in vivo. The possible relevance of these findings to augmented ammoniagenesis following acid challenge is discussed.Materiab and Methods. Albino rats g ; Zivic-Miller, Allison Park, Pa.) were fed water and rat chow ad libitum. 24 hr prior to study, water was replaced with 1.5% NaHC03 to insure that no rat was acidotic at the time of study.For in vitro studies, rats were sacrificed and the kidneys were rapidly placed in iced saline. Within 15 min, slices were cut on a Supported by a grant from the National Institute of Health AM 11525.Stadie-Riggs microtome. Only the first slice from the outer surface of the kidney was used. Each slice was halved, half was used as control, the other half was incubated in malonate or fluorocitrate (K and K Chemical). Eight slice pairs were used in the studies with malonate and 10 slice pairs with fluorocitrate. In the studies concerned with citrate gluconeogenesis (Fig. 2 ) , six slices were halved, half was placed in citrate (10 m M ) and the other half in citrate (10 mM) and barium fluorocitrate (1.0 mM). When glutamate or citrate was used as substrate the concentration was 10.0 mM. Inhibitors were added in the concentrations indicated in the text. Medium used for all experiments was a modified Krebs-Ringers solution buffered with 10 d phosphate to a pH 7.4.For in vivo studies, rats were injected with 1.2 ml of 1.0 M malonate/100 g of rat ( 7 ) or 1 ml of 4 M barium fluorocitrate/lOO g of body wt. Control rats received an equivalent volume of saline intraperitoneally. To assure diuresis all rats received 5.0 ml of H2O through a gastric tube. At the end of 4 hr, the bladder urine was evacuated by ether stim'ulation. Urines were collected under toluene; and pH, volume, and ammonia concentration were measured.Ammonia and glucose were estimated by methods previously described (8). Citrate was measured by a citrate lyase technique (9). Statistics are by pair or group analysis using the Student's t test.Results. In Vitro. In the presence of 10 mM malonate, ammoniagenesis from gluta...

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To determine the areas of rat kidney which increase ammoniagenesis in response to acid challenge, ammonia production was followed in vitro in slices from the cortex, outer stripe of outer medulla, inner stripe of the outer medulla, and inner medulla (papilla). When the medium pH was lowered to pH 7.0, increased production of ammonia occurred only in the cortical tissue. When kidney slices were removed from chronically acidotic rats, ammonia production from glutamate increased in the cortex, and outer and inner stripe of the outer medulla. Despite small increases in ammonia production from glutamate in the outer medulla of chronically acidotic rats, it is concluded that the major adaptive increases in ammoniagenesis in response to acid challenge take place in the renal cortex.

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