Sex differences in the subunits of glutathione-S-transferase isoenzyme from rat and human kidney

Glutathione-S-transferase (GST) isoenzymes were purified from cytosolic preparations from kidneys of male and female rats and kidney cortical specimens from 2 male and 1 female human subjects. GST isoenzyme expression was analyzed by SDS-PAGE, measurement of catalytic activities with specific substrates and determination of their subunits by ELISA and Western blotting using specific antibodies. GST from female rat kidneys showed a preponderance of subunits 3 and 4; levels of these isoenzymes were 3-4 times greater in females than in males. Levels of subunits 1 and 2 were 1.5-2 times greater in the male rat kidneys. Additional minor bands at 24 and 22 kD were observed in GST preparations from both male and female rat kidneys while a band at 25.3 kD was observed only in the male rat kidney. These bands did not react with antibodies to GST 1-1, GST 2-2 or GST 3-4. Both male and female human kidney samples contained GST isoenzymes comparable to the near-neutral (25-5 kD) and basic forms (25 kD) of GSTs found in human liver. In addition a 28-kD band was present in GST preparations from both male and female human kidneys. Additional bands at 29 and 25.2 kD were present only in male human kidneys. Both the kidney cytosol and the total GSTs prepared from female rats shared 2- to 4-fold greater activity with 1,2-dichloro-4-nitrobenzene, ethacrynic acid and trans-4-phenyl-3-buten-2-one than those from males. The measurement of specific subunit amounts by ELISA were in agreement with these results.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of histamine in the rat kidney during pregnancy and development

Summary An antiserum against conjugated histamine and two oligonucleotide probes that detect the mRNA encoding L-histidine decarboxylase (HDC) involved in histamine synthesis were used to study the appearance of histamine and its location in the kidneys of fetal, newborn and young postnatal rats and in the kidneys of pregnant rats. On embryonic days 16 and 18 (E16 and E18), some HA-immunoreactive (HA-ir) cells were found within the largest S-shaped bodies. Histamine was found to appear rapidly between the 18th and 20th embryonic days in the convoluted tubules of the kidneys. On postnatal day 0 (P0), the distal convoluted tubules and collecting ducts exhibited bright fluorescence, the intensity of which decreased quickly so that it was faint on day P4 and absent at later stages. In kidneys of pregnant rats HA-ir was found in the epithelium of both the Bowman's capsule, collecting ducts and in a few cells within the tubules. Nonuniform HA-ir was also detected within glomeruli. No evidence for the presence of L-histidine decarboxylase mRNA in kidneys of fetuses or pregnant rats was seen. It is concluded that distinct structures in the developing rat kidney contain histamine during a period around birth from day E20 to day P4. In the pregnant rat, the epithelium that is in direct contact with the urine flow is immunoreactive for histamine from day 16 to 20 of pregnancy. The results suggest that histamine is not synthesized locally in the kidneys but rather originates from other tissues.     

Distribution of actin bundles in Bowman's capsule of rat kidney

In this study we define the distribution of actin bundle arrangement in Bowman's capsule of rat renal corpuscles. Parietal cells of Bowman's capsule were examined by conventional light microscopy, electron microscopy and confocal microscopy. Within each parietal cell individual actin bundles are arranged in a parallel fashion running the length of the cell. Computer reconstructions obtained using confocal microscopy clearly show the lengths of actin bundles to be arranged, on a capsule level, end-to-end, at angles and perpendicular to bundles in adjacent cells. The bundles stain positively for non-muscle myosin and vinculin. The presence and arrangement of actin bundles in parietal cells is consistent with a role in reinforcing capsule structure.     

Morphology of rat kidney and thymus after native and antibody-coupled cyclosporin A application (Reduced toxicity of targeted drug)

This study compares the toxic effects of native cyclosporin A (CyA) with those of targeted CyA that is conjugated with the anti-rat-thymocyte antibody of rabbit originvia the N-(2-hydroxypropyl)methacrylamide (HPMA) carrier bearing digestible, reactive oligopeptide side chains. Ten toxic doses of native CyA (50 mg/kg i.p.) given to young adult rats in the course of 14 d produced a severe renal lesion—diffuse microvacuolization of the proximal tubules in the deep cortex, and hypergranulation of juxtaglomerular regions. Severe atrophy of the thymic medulla was documented by morphometry. In the cortex the epithelial reticular (but not deep interdigitating) cells showed ultrastructural signs of severe degeneration and lysis. The immature CD4⁺8⁺ double-positive cortical lymphocytes were preserved whereas, the single-positive medullary thymocytes were greatly depleted; there was also a restriction of MHC class II antigen expression in the medulla. The number of medullary B cells was increased. The cytokeratin net was focally shrunken in the cortex and almost negative in the medulla, with loss of Hassall's corpuscles. After ten corresponding doses of antibody-targeted conjugated CyA no damage to the renal tubules and arterioles appeared and the antiGBM or immune-complex deposition was absent. The thymus had a normal medulla with numerous mature thymocytes and the cortical epithelial reticulum remained well preserved. Thus, the main toxic effects of CyA could be eliminated by targeting. The T-cell-targeted drug was tested for preserved immunosuppressive properties and non-toxic character of HPMA copolymer carrier.     

Distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney

Summary In the present study we have investigated the distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney. The distribution of IGF-I mRNA was investigated using a simple and sensitive non-radioactive in situ hybridisation technique based on probe labelling with digoxigenin labelled-UTP followed by detection with conventional immunocytochemical techniques. IGF-I mRNA was found predominantly in medullary collecting ducts and sparsely in cortical collecting duct cells. In addition IGF-I mRNA was expressed in scattered proximal tubular cells in the cortex and in cells confined to the glomerular tuft. IGF-I binding sites were studied using radiolabelled IGF-I and conventional autoradiographical techniques on tissue sections. It was found that IGF-I binding sites were widely distributed throughout the entire kidney and that the specific binding was highest in the inner medulla. These findings add further complexity to the understanding of IGF-I production and action on renal structures.     

A novel cytochrome P450 3A isoenzyme in rat intestinal microsomes

PCR with several pairs of primers facilitates screening for new isoenzymes among highly homologous cytochrome P450s (CYPs). Combinations of two pairs of primers, which amplify N- and C-terminal coding sequences of either CYP3A1/CYP3A23 or CYP3A2 detected the presence of a previously unrecognized CYP3A in enterocyte microsomes isolated from rats. PCR, Northern blot, and immunoblotting with specific antibodies indicated that this isoenzyme is clearly distinguishable from CYP3A1, 3A23 or 3A2. Sequencing of a 285 bp coding fragment of this gene revealed 97% similarity with rat olfactory CYP3A9 (P450olf3).     

Tissue-specific uncouplers of oxidative phosphorylation in mitochondria from rat heart, kidney, thymus and lung]

The influence of nonmicrosomal cytoplasm fraction from the rat kidney, heart, thymus and lungs on the oxidative phosphorylation of mitochondria (M) of the same tissues and from the ones of the liver and brain was studied. The existence of the tissue-specific uncouplers of the M oxidative phosphorylation was shown in the cross experiments; they were similar to that in the rat liver revealed earlier. A possibility of these regulators participation in the initiation of the M enzymatic degradation process through the activation of phospholipase A when calcium ions leave the M is discussed. The activation under these conditions of the DNA-ase 1, associated with the M membrane, is suggested.     

Distribution of different cell types within the rat thymus in the neonatal period of life

Summary This study attempted to define reciprocal positions of cell types within the thymus. Random or non-random contacts between specific cell types were analyzed by means of graph theory. For analysis, thymus blocks were sectioned serially and, then, thymus cells were categorized into types, based on morphological criteria. The distribution of individual cell types within the cortex, cortico-medullary zone and medulla was presented in form of a map. In the analysis, three types of epithelial cell, characteristic of each thymus zone, macrophages, Langerhans-like cells and lymphocytes were found in non-random relations to one another. Moreover, characteristic groups of cells associated with one another were also demonstrated.This study was supported by the Polish Academy of Sciences     

Androgen-regulated ornithine decarboxylase mRNAs of mouse kidney

Ornithine decarboxylase, the first enzyme of the polyamine biosynthetic pathway, is induced by androgens in the mouse kidney. We have isolated from a kidney cDNA clone bank two plasmids, pODC1440 and pODC934 , that contain different cDNA inserts corresponding to ornithine decarboxylase mRNA. Identification was based upon the ability of plasmid-specific mRNAs to encode a 53,000-dalton polypeptide that reacts with antibody to purified mouse kidney ornithine decarboxylase. Plasmid pODC1440 hybridizes predominantly to a mRNA that is 2.1 kilobases (kb) long and is induced about 20-fold in the kidneys of female mice treated with testosterone. Plasmid pODC934 hybridizes to mRNAs of lengths 2.2 and 2.6 kb, which are induced about 8-fold by testosterone. There are probably no more than 1-2 copies of the pODC1440 -specific sequence in the mouse genome, while there may be as many as 12 copies of the pODC934 -specific sequence. That the two plasmids correspond to different ornithine decarboxylase mRNAs is suggested by two observations. First, several mouse strains express normal levels of pODC934 -specific RNA and little or no pODC1440 -specific RNA; furthermore, pODC934 -specific RNA is expressed in several tissues while pODC1440 -specific RNA is kidney-specific. Thus, androgen-mediated stimulation of kidney ornithine decarboxylase activity levels involves alterations in the concentrations of at least two distinct mRNAs.     

Distribution of endothelin receptor subtypes ETA and ETB in the rat kidney.

The endothelin (ET) receptor system is markedly involved in the regulation of renal function under both physiological and pathophysiological conditions. The present study determined the detailed cellular localization of both ET receptor subtypes, ET(A) and ET(B), in the vascular and tubular system of the rat kidney by immunofluorescence microscopy. In the vascular system we observed both ET(A) and ET(B) receptors in the media of interlobular arteries and afferent and efferent arterioles. In interlobar and arcuate arteries, only ET(A) receptors were present on vascular smooth muscle cells. ET(B) receptor immunoreactivity was sparse on endothelial cells of renal arteries, whereas there was strong labeling of peritubular and glomerular capillaries as well as vasa recta endothelium. ET(A) receptors were evident on glomerular mesangial cells and pericytes of descending vasa recta bundles. In the renal tubular system, ET(B) receptors were located in epithelial cells of proximal tubules and inner medullary collecting ducts, whereas ET(A) receptors were found in distal tubules and cortical collecting ducts. Distribution of ET(A) and ET(B) receptors in the vascular and tubular system of the rat kidney reported in the present study supports the concept that both ET receptor subtypes cooperate in mediating renal cortical vasoconstriction but exert differential and partially antagonistic effects on renal medullary function.     

Distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney.

In the present study we have investigated the distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney. The distribution of IGF-I mRNA was investigated using a simple and sensitive non-radioactive in situ hybridisation technique based on probe labelling with digoxigenin labelled-UTP followed by detection with conventional immunocytochemical techniques. IGF-I mRNA was found predominantly in medullary collecting ducts and sparsely in cortical collecting duct cells. In addition IGF-I mRNA was expressed in scattered proximal tubular cells in the cortex and in cells confined to the glomerular tuft. IGF-I binding sites were studied using radiolabelled IGF-I and conventional autoradiographical techniques on tissue sections. It was found that IGF-I binding sites were widely distributed throughout the entire kidney and that the specific binding was highest in the inner medulla. These findings add further complexity to the understanding of IGF-I production and action on renal structures.     

Cyclosporine A and purinergic receptors in rat kidney

Previous reports have demonstrated that Cyclosporine A (CyA) chronically administered induces an increase in adenosine plasma concentration by inhibiting adenosine uptake by red blood cells (RBC). We hypothesized that this effect may modulate, by a down regulation, the mRNA expression of adenosine receptors in rat kidney. Since high blood pressure (HBP) is a classical side effect of CyA treatment, nicardipine, a dihydropyridine calcium channel blocker, is often associated with CyA in treatment. To distinguish between the effects of CyA-induced HBP and the effects of CyA by itself, we have evaluated the effects of CyA and/or nicardipine on the mRNA expression of A1 and A2a adenosine receptors. The study was performed on five groups of rats (n= 8) receiving during 21 days either serum saline (0.5 ml i.p), CyA (12 mg/kg/day, i.p), nicardipine (1.2 mg/kg i.p) or nicardipine + CyA. The last (or fifth) group was injected with vehicle (0.5 ml i.p). Blood samples for adenosine assay were collected in the renal artery at day 21, just before the rat kidneys were removed for quantitation of adenosine A1 and A2a mRNA concentration by RT-PCR. We make two conclusions :i) Nicardipine induces a decrease in mRNA expression of A1 but not of A2a adenosine receptors. However, because nicardipine lowered both blood pressure and A1 mRNA expression, it is not possible to conclude if A1 mRNA decrease is implicated in the nicardipine effects on blood pressure.ii) CyA induces an increase in renal artery adenosine concentration and a decrease in mRNA expression of A1 and A2a adenosine receptors.     

Distribution of some hydrolases in the rat kidney (author's transl)

Most of the available histochemical methods and techniques (azodye, metal salt and indigogenic methods, cryostat, free-floating and lyophilized section techniques) and different modifications of these methods (different substrate concentrations, pH, temperature, incubation time e.g.) were applied to study the distribution of acid phosphatase (AcPB = after Barka and Anderson; AcPG = after Gomori), beta-glucuronidase (beta-Glu), aryl sulfatase (AS), beta-N-acetylglucosaminidase (NAG), acid 5'-nucleotidase (a5-Nucl), non-specific esterase (NE) and alkaline phosphatase (AlP) in the kidneys of rats of both sexes. The optimal conditions for the demonstration of these enzymes were established. As most important proved: the incubation of free-floating sections cut from "standard"-fixed (2 h in formol-calcium continued for another 18-22 h in the same fixative plus 0.88 M sucrose at 4 degrees C) kidney slices - only for AcPB and NE material fixed after Holt had to be used; the incubation for AlP and NE at 4 degrees C; final pH of the incubation medium for AcPB 5.5, AcPG 5.0 and NE 6.5; the use of Fast Garnet GBC Salt as coupler in the NE azo-dye reaction. Sex differences and for the female rats an increased activity during oestrus were established for all hydrolases studied. In particular the following results were obtained: AcPB, a5-Nucl and A1P are more intensive in male and AcPG in female S1 segments of the juxtamedullary nephrons in relation to the nephrons of the other parts of the cortex. In the medullary rays the NE and the a5-Nucl show a higher activity in the S2 segments of female rats demonstrate a more intensive activity for NAG and NE. This is true for AcPG and A1P in male rats. In the inner medulla a stronger beta-Glu activity in male rats and a stronger NAG activity in female rats is observed. The AcPB activity of the cortical distal tubules is higher in male rats.