Arachidonic acid epoxygenase. Stereochemical analysis of the endogenous epoxyeicosatrienoic acids of human kidney cortex.

Mass spectral and chromatographic analysis demonstrates the presence of 14,15-, 11,12- and 8,9-epoxyeicosatrienoic acids (44%, 33% and 23% of the total, respectively) in human kidney cortex. Chiral analysis of the human renal epoxyeicosatrienoic acids shows the formation of 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids in a 1:1, 4:1 and 2:1 ratio of antipodes, respectively. These results demonstrate the biosynthetic origin of the human kidney 11,12- and 14,15-epoxyeicosatrienoic acids and suggest a role for renal cytochrome P-450 in the bioactivation of endogenous pools of arachidonic acid.

Histochemical and immunocytochemical evaluation of copper and metallothionein in the liver and kidney of copper-loaded rats.

Histochemical methods do not always show a good correlation with analytical measurement of copper content and consequently immunoreactive staining techniques for metallothionein (MT) have recently been employed for the differential diagnosis of copper-associated diseases. This study compares histochemical with immunocytochemical methods for the assessment of copper status. Male rats were fed a high copper (1 g/kg) diet for 16 weeks and killed sequentially during this period. The livers and kidneys were analysed for copper and zinc (atomic absorption spectrophotometry), and sections were stained with rubeanic acid and rhodanine for copper and for immunoreactive MT using the DNP localization system. Immunoreactive stains for MT corresponded better with copper content than histochemical stains and were more sensitive, albeit less selective, indicators of copper accumulation. Moreover, major differences in intracellular staining were apparent between the two methods, attributed to differences in copper binding and microcompartmentalization of metal.

Nephritogenic glycoprotein. XIV. A simple isolation method of nephritogenoside.

Normal rat renal cortex contains three renal disease-inducing substances, that is, nephritogenoside, FX1A and immunogenoside (a novel renal glycoprotein that has the biological activity to induce only active Heymann nephritis). When renal cortex homogenate was treated with a detergent, Triton X-100, nephritogenoside was not eluted in the supernatant in which massive doses of FX1A and immunogenoside were detected. Thus, we searched for nephritogenoside in the precipitate. The precipitate was solubilized with trypsin, and nephritogenoside was easily and effectively isolated through columns of DEAE-cellulose followed by Bio-Gel A-1.5 m. The purified sample thus obtained is only composed of nephritogenoside, and contains neither FX1A nor immunogenoside. The yield of purified nephritogenoside was 3.8 mg (starting from 150 rats), which is about 5-6 times the recovery of the previous methodology. This newly developed simple method may be useful for the isolation of purified nephritogenoside. The chemical and immunologic properties of the purified sample prepared by the new method corresponded well with those of the nephritogenoside which was obtained by the previous methodology after exhaustive digestions with three proteolytic enzymes from homologous renal cortex.

Immunocytochemical demonstration of vasopressin binding in rat kidney.

We investigated the immunoperoxidase demonstration of vasopressin (VSP) bound to paraffin-embedded sections of rat kidney and the effects of various fixatives. Slices of rat kidney from normal and 4-day water-deprived rats were incubated with 10(-7) M VSP, fixed, and embedded in paraffin. Hydrated sections of these tissues were again incubated with 10(-7) M VSP or 10(-7) M VSP and 10(-5) M oxytocin (OXY). VSP bound to the sections was demonstrated using rabbit anti-Arg8 VSP antiserum and peroxidase-labeled second antibody. In sections of kidney from both normal and water-deprived rats, immunoperoxidase labeling was most intense in the renal papilla and was restricted to the cells of the ducts of Bellini and loops of Henle. In the medulla, the collecting ducts and medullary thick ascending limbs of Henle were moderately stained. In the normal kidney sections there was no staining of the proximal tubules, distal convoluted tubules (DCT), and only slight staining of the cortical collecting ducts (CCD). However, in the water-deprived rats there was a considerable increase in the staining of the DCT and CCD. Simultaneous incubation in OXY and VSP resulted in reduced immunoperoxidase labeling of the tubules. Omission of VSP incubation led to a similar decrease in stain intensity, indicating a specificity for the sites of VSP binding. This technique allows the identification of cells responsible for the binding of VSP in the kidney.

The effect of oral calcium load or verapamil on gentamicin-induced nephrotoxicity.

Several investigators have reported recently that in rats, oral calcium load is associated with a marked amelioration in gentamicin-induced renal failure. In contrast to these reports, using the same animal model, we could not observe calcium-induced moderation in gentamicin nephrotoxicity as reflected by either urea or creatinine serum concentration or by various renal cortical intracellular enzymatic activities. Similarly, verapamil, a calcium channel blocker, had no effect on the degree of renal failure in these animals. We conclude that manipulation of calcium diet may not be uniformly effective in reducing gentamicin nephrotoxicity. Additional nutritional factors may play a crucial role in achieving the amelioration of this model of toxic nephropathy.

Effect of dietary taurine on renal taurine transport by proximal tubule brush border membrane vesicles in the kitten.

Renal adaptation of the kitten to altered dietary taurine intake was assessed using proximal tubule brush border membrane (BBM) vesicles. Three groups of kittens were adapted to purified diets containing 43.5% soy protein that were either taurine-free (OT) or contained 0.15% taurine (NT) or 1.0% taurine (HT). The plasma taurine concentration of the kittens fed OT decreased from 104 +/- 25 microM to 16 +/- 5 microM and 1.7 +/- 0.5 microM in 1 and 6 wk, respectively. Feeding HT increased plasma taurine concentration to 350 +/- 116 microM in 1 wk. Compared to NT, taurine accumulation by BBM vesicles was significantly elevated after 4 wk of feeding OT and decreased after 2 wk or less of feeding HT (P less than 0.05). Maximum renal adaptation occurred by 6 wk of feeding OT (206% increase in taurine uptake/15 s compared to NT) and by 2 wk or less of feeding HT (43% decrease in taurine uptake/15 s compared to NT). Evaluation of transport kinetics using renal cortex from groups of four kittens (16 determinations) fed NT, OT (12 wk) or HT (10 wk) revealed a Vmax of 55 +/- 10, 123 +/- 24 or 39 +/- 7 pmol.mg protein-1.10 s-1 and a Km of 32 +/- 7, 16 +/- 2 or 37 +/- 8 microM, respectively. The differences in Vmax and Km were significant between NT and OT (P less than 0.05), but not significant between NT and HT (P greater than 0.05). Our results suggest that renal adaptation of the kitten to changes in dietary taurine occurs with modifications of both Vmax and Km of the taurine transport system.

Cadmium exposure in Denmark. Based on analyses of liver and kidney tissues.

To determine the cadmium exposure level in the Danish population, tissue samples of kidney and liver were collected over the period 1981 to 1987 at the Institutes of Forensic Medicine in Copenhagen, Odense and Aarhus. A total of 795 samples were collected, of which 143 were selected for analysis using, the criteria sudden violent death (accident, homicide or suicide). Cadmium concentrations in kidney cortex increased by age to a maximum of approximately 22 micrograms/g/g (w/w) in the age group 45-55 years and decreased in the older age group, while liver concentrations tended to increase throughout the entire lifespan. Cadmium concentrations in kidney cortex and liver were found significantly correlated. The findings are in good agreement with internationally published data, but lower than previously reported Danish concentration levels. The reason for this difference is discussed.

Characterization of two possible forms of type IV collagen from human kidney cortex.

Type IV collagen was isolated from human kidney cortex according to the purification of mouse kidney type IV collagen (MKIVC) with minor modifications as described previously [Oikawa, T., et al. (1986) Chem. Pharm. Bull. 34, 789-797]. Chromatography of human kidney type IV collagen (HKIVC) on a column of DEAE-cellulose resulted in its separation into two fractions, i.e., one (HKIVC-1) passed through and the other (HKIVC-2) adsorbed to the column, similar to MKIVC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Bio-Gel A-5m chromatography revealed that both HKIVC-1 and -2 differed from each other in the ratios of major and minor components. Amino acid analyses also demonstrated that there was a significant difference in the contents of several amino acid residues between both type IV collagens and their 80-kDa components, one of the major ones. These results indicate the possibility that there exist two forms of type IV collagen in human kidney.

Identification and characterization of parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding.

Parathyroid hormone (PTH) receptors have been described in renal tissue from several species, but not in the rat. In this study, radioligand binding techniques were used to identify and characterize PTH receptors in rat kidney cortical membranes. The sulfur-free PTH analog [Nle8,18Tyr34]bovine PTH-(1-34)amide was iodinated using the iodogen method. This ligand was suitable for use in identifying PTH receptors in canine renal membranes, but not rat renal membranes. Synthetic, unsubstituted rat PTH-(1-34) was iodinated using the milder, lactoperoxidase technique and was purified by HPLC on a C8 column. [125I]rat PTH-(1-34) bound rapidly to both rat and dog renal membranes. At 22 degrees C reaction reached steady state within 20 minutes, and this level was maintained for at least 3 h. Specific binding was routinely greater than 90% for rat kidney and greater than 95% for dog kidney. Similar results were obtained at 4 degrees C with a longer time required to attain steady state (approximately 45 minutes). Binding was reversible as demonstrated by dissociation of bound ligand after either infinite dilution or displacement with excess nonradioactive PTH. Binding was saturable and of high affinity (rat kidney: Bmax = 2.3 pmol/mg protein, Kd = 3.1 nM, dog kidney: Bmax = 2.1 pmol/mg protein, Kd = 3.7 nM). Rat renal cortical adenylate cyclase activity was stimulated by rat PTH in a dose-dependent manner with an EC50 of 4 nM, a value in good agreement with the binding data. This study demonstrates the feasibility of identifying and characterizing parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding techniques.

Identification of P-glycoprotein in renal brush border membranes.

A monoclonal antibody (C219) that recognizes the P-glycoprotein (Mr = 170,000) in plasma membranes of multidrug-resistant Chinese hamster ovary (CHO) cell lines was used to assay renal brush border membrane (BBM) and basolateral membrane (BLM) fractions for the presence of a cross-reactive polypeptide. The C219 antibody bound to a 155,000 dalton protein in immunoblots of rat BBM but not BLM proteins resolved by sodium dodecyl sulfate gel electrophoresis. The corresponding human kidney BBM and dog kidney BBM proteins had molecular weights of 170,000 and 160,000 respectively. The glycoprotein nature of the renal protein was shown by its sensitivity to N-glycanase treatment which reduced the apparent molecular weight of the dog protein to 120,000. In addition, dog P-glycoprotein could be bound to and eluted from immobilized wheat germ agglutinin. The molecular weight, antibody crossreactivity, glycosidase sensitivity and lectin binding show that this protein is a normal kidney analogue of the P-glycoprotein induced in multidrug resistant cell lines.

Effects of sorbinil on glomerular structure and function in long-term-diabetic rats.

We investigated the role of the aldose reductase pathway in the pathogenesis of the nephropathy of rats with sever (non-insulin-treated) streptozocin-induced diabetes of 6 mo duration. The initial experiment included four groups of rats: diabetic and control animals on a 20% protein diet, which were untreated or treated with sorbinil (an aldose reductase inhibitor). Food intake was increased by diabetes but was uninfluenced by sorbinil, whereas urinary urea nitrogen excretion was increased and body weight was decreased by both variables. Glomerular basement membrane (GBM) width was increased by diabetes and decreased by sorbinil. No other structural changes were noted. We speculated that sorbinil could have slowed the abnormal rate of GBM thickening in diabetic rats and the normal increase in GBM width in control rats by inducing a mild catabolic state. The second experiment also involved four groups of rats: diabetic and control animals on a 50% protein diet, which were untreated or treated with sorbinil. In these studies, diabetes was again associated with reduced body weight, but sorbinil had no influence on urinary urea nitrogen. Urinary albumin excretion, which was increased by diabetes, was not affected by sorbinil. GBM width was increased by diabetes, but in contrast to animals on 20% protein diets, the animals on 50% protein diets and treated with sorbinil did not have reduced GBM widths. Mesangial volume fraction was greater in diabetic animals than in controls, and sorbinil largely prevented mesangial expansion in them. Surprisingly, the control animals on the 50% protein diet and given sorbinil had increased mesangial volume fraction compared with control rats on the same diet not given the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Extrarenal Wilms' tumor: an analysis of four cases.

During a review of Wilms' tumor, four located external to the kidney were identified. Patient age ranged from 7 months to 4 years; three were female. One neoplasm was located in the parametrial connective tissue to the left of the uterus; both kidneys were radiographically normal. Three neoplasms were located in the right retroperitoneum adherent to the surface of the ipsilateral kidney, but separated from the parenchyma by a thick fibrous capsule. Two were attached to the upper pole, while the third was attached to the midportion of the kidney. Radiologic studies showed displacement of all three kidneys, but intravenous pyelogram (IVP) revealed no calyceal distortion. All four neoplasms were favorable histology Wilms' tumor: one was monophasic epithelial type, one was monophasic blastemal type, and two had a mixture of stromal, epithelial, and blastemal tissue. No teratomatous elements were present. Immunoperoxidase staining for cytokeratin (AE-1, AE-3, CAM 5.2), vimentin, and epithelial membrane antigen (EMA) showed the strongest focal positive staining of tubular structures with CAM 5.2, and slight staining with EMA. Staining reaction to vimentin was variable, but negative in most areas. Three tumors extracted from paraffin were diploid by quantitative flow cytometric DNA analysis; in one case, flow cytometry could not be performed. Clinical follow-up from 2 years to 6 years showed all children to be alive without evidence of disease. Based on the similarity to conventional renal Wilms' tumor, these findings support the hypothesis of displaced mesonephric/metanephric rests for the origin of extrarenal Wilms' tumor.

Purification and reconstitution of chloride channels from kidney and trachea.

Chloride channels mediate absorption and secretion of fluid in epithelia, and the regulation of these channels is now known to be defective in cystic fibrosis. Indanyl-oxyacetic acid 94 (IAA-94) is a high-affinity ligand for the chloride channel, and an affinity resin based on that structure was developed. Solubilized proteins from kidney and trachea membranes were applied to the affinity matrix, and four proteins with apparent molecular masses of 97, 64, 40, and 27 kilodaltons were eluted from the column by excess IAA-94. A potential-dependent 36Cl- uptake was observed after reconstituting these proteins into liposomes. Three types of chloride channels with single-channel conductances of 26, 100, and 400 picosiemens were observed after fusion of these liposomes with planar lipid bilayers. Similar types of chloride channels have been observed in epithelia.

Renal proton magnetic resonance in experimental acute renal failure in rats.

Cortical, medullary and papillary T1 and T2 water proton relaxation times were measured at 37 degrees C, 20 MHz. The measurements were made using kidneys from rats affected by many forms of experimental acute renal failure (ARF), namely acute hemorrhagic hypovolemia, angiotensin II administration, antidiuretic hormone (ADH) administration, glycerol, and other nephrotoxins (gentamicin, cisplatinum, cyclosporine), renal artery occlusion for different periods of time, and ureteral ligation. From the T1 and PW (percent tissue water content) the bound water (FB) and HF (percent water bound/g solid) were calculated according to a fast proton diffusion model. In most experimental models studied, the experiments were repeated following paramagnetic enhancement with GdDTPA administration (70 mmol/kg BW). By profiling the deviations from normal, it was possible to differentiate the ischemic (shortened T1, prolonged T2), obstructive (very high T1 and T2 in both cortex and medulla) and nephrotoxic (prolonged T2) forms of ARF. Significant changes in free/bound water compartments occurred, though their biological significance is unknown. T1 and T2 ratios before and after paramagnetic enhancement correlated well with estimates of glomerular filtration rate. In the first minutes following acute hemorrhagic hypovolemia, the intrarenal water distribution remained unchanged. After GdDTPA significant water proton T1 and T2 changes characterized the immediate posthemorrhagic state similar to the effect of ADH.

Interactions between aminoglycosides and phospholipids using liposomes: a possible mechanism of nephrotoxicity.

Interactions of aminoglycosides with phospholipids were estimated by the increase in turbidity of liposomes consisting of various phospholipids. The turbidity of liposomes containing negatively charged phospholipids was increased by gentamicin, the highest increase in turbidity being observed for phosphatidylinositol-4,5-diphosphate-containing liposomes. The extent of turbidity was dependent on the concentration of acidic phospholipid in the liposomal membrane as well as the number of amino groups of the aminoglycosides. The release of glucose from glucose-entrapped liposomes depended on the concentration of gentamicin. The turbidity of liposomes containing lipids extracted from rat renal cortex was also increased by aminoglycosides depending on the number of amino groups. From electron microscopic observations, the increase in turbidity of liposome suspensions was caused by liposome fusion.

Postmortem metabolic indices of antemortem adrenergic stimulation.

Tissue lactate concentration has been reported to be a useful postmortem indicator of antemortem awareness of mortal danger. The purpose of this study was to determine further whether selected tissue metabolites could be used as postmortem markers of antemortem adrenergic stress. Sprague-Dawley albino rats were anesthetized with pentobarbital and then injected with 2.0 mg kg-1 i.p. epinephrine hydrochloride to induce experimentally a severe sympathetic response that may be associated with the awareness of mortal danger; 20 min after the injection of epinephrine, when the metabolic response was at its peak, the animals were killed by exsanguination. Samples of the following tissues were removed immediately prior to death (0 h) and 48 h postmortem: soleus, plantaris, kidney medulla, kidney cortex, liver, and heart. These samples were analyzed for glycogen, lactate, ATP, creatine phosphate, pH, and total protein concentration. Significant differences in lactate concentration were observed in all tissues except soleus at 0 h in the epinephrine-injected animals. Specific tissues also had significant reductions in glycogen, ATP, and creatine phosphate concentrations at 0 h. At 48 h postmortem, however, only the liver and soleus lactate concentrations were significantly different from the 48-h control samples. It is unlikely that these small differences found in some tissues at 48 h postmortem would be detected in an uncontrolled accident situation. We concluded from these findings that these selected tissue metabolites are not useful as long-term postmortem indicators of antemortem adrenergically induced hypermetabolism.

Tissue specificity of chromatin glycoproteins recognized by concanavalin A.

Chromatin glycoproteins recognized by Concanavalin A have been isolated from pig liver, kidney and heart by the use of immobilized lectin. Two groups of proteins differing in affinity for DNA have been analysed. Glycoproteins are mainly present in the group of proteins which are tightly bound to DNA. Mono and bidimensional electrophoretic patterns of total tightly bound proteins reveal a similarity among the three organs examined, while the corresponding patterns of the glycoproteins are typical for each organ. The tissue specificity of chromatin glycoproteins, together with their capability to interact not only with DNA but possibly also with other nuclear components, suggest a role for these proteins in the mechanism of genome expression.

Mercury accumulation in tissues from dental staff and controls in relation to exposure.

Samples, mainly from occipital cortex and pituitary gland, but also from rental cortex, olfactory bulbs, thyroid gland and liver were collected from autopsies of 8 dental staff cases and 27 controls. These samples were analysed for total mercury content using radiochemical neutron activation analyses. The results revealed high mercury concentrations (median 815, range 135-4,040 micrograms Hg/kg wet weight) in pituitaries from the dental staff cases compared to controls (N = 23, median 23 range 6-1, 170 micrograms Hg/kg). In occipital cortex, the cases had a median of 17, range of 4-300 micrograms Hg/kg and the controls (N = 20) had a median of 10, range 2-29 micrograms Hg/kg. A few samples from olfactory bulbs show low mercury concentrations for both cases and controls. Renal cortex was analysed from three cases and contained clearly higher concentrations (945, 1,545, 2,110 micrograms Hg/kg) compared to controls (N = 12, median 180, range 21-810 micrograms Hg/kg). There is no control material for the other analysed samples, but one thyroid sample had an extremely high concentration of 28,000 micrograms Hg/kg.