Normal Rabbit Kidney Research Articles

The ultrastructure of the macula densa during altered sodium intake. A morphometric study of the macula densa in the rabbit nephron.

Cells of the macula densa from sodium-depleted, sodium-loaded and normal rabbit kidneys were investigated morphometrically by light and electron microscopy. The total macula densa volume constituted 0.14 per cent of the cortical volume and was unchanged during altered sodium balance. Ultrastructurally the macula densa cells appeared similar at the three different levels of sodium intake investigated. However, the relative volume of Golgi apparatus in the cells of the macula densa decreased from the normal value of 1.54 per cent of the cytoplasmic volume to 1.25 per cent during sodium-load, indicating that functional changes take place in the cells of the macula densa under these conditions. The volume of the Golgi apparatus was unchanged in the sodium-depleted group. The volume of the cytoplasm, the nuclei and the intercellular space which normally constitute 56 per cent, 28 per cent and 15 per cent of the total volume of the macula densa, respectively, were unchanged during sodium-load and sodium-depletion. The mitochondria which normally constitute 25 per cent of the cytoplasmic volume were similarly unchanged.

Contribution of thromboxane to renal resistance changes in the isolated perfused hydronephrotic rabbit kidney.

Thromboxane A2 is not produced in normal rabbit kidneys, but its synthesis is induced in numerous renal pathological states. The presence of this potent vasoconstrictor could readily compromise renal hemodynamics. We found that the thromboxane synthetase inhibitor, OKY-1581 (sodium-3-[4,3-pyridylmethyl)phenyl]-2-methylacrylate) is effective in the perfused hydronephrotic kidney in selectively inhibiting thromboxane production without altering prostaglandin E2 or prostacyclin release. The vasoactive peptides bradykinin and angiotensin cause the hydronephrotic kidney to produce thromboxane A2, which results in a profound renal vasoconstriction which is reversed by pretreatment with OKY-1581. Thus, OKY-1581 provides a powerful tool which can be used to assess the participation of thromboxane in pathophysiological states and to ascertain the therapeutic potential of thromboxane synthetase inhibitors in numerous disease states.

Regional synthesis of monohydroxy eicosanoids by the kidney.

Arachidonic acid was incubated with 10,000 X g and 100,000 X g supernatants prepared from the medullae of hydronephrotic, normal, and contralateral rabbit kidneys. Metabolites whose production was calcium dependent but not inhibited by indomethacin were identified by thin layer chromatography. Preparative separation was carried out by silicic acid high pressure liquid chromatography. The methyl ester trimethylsilyl derivatives were prepared and examined by gas chromatography-mass spectrometry. The mass spectra matched previously published mass spectra for 12-hydroxy-5,8,10,14-eicosatetraenoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid. The renal cortex does not metabolize arachidonic acid through the lipoxygenase pathway. Thus, there is regional metabolism of arachidonate to monohydroxy eicosanoids in the kidney.

Pyelorenal Backflow in Normal and Ischemic Rabbit Kidneys

Pyelorenal backflow during retrograde pyelography was studied in 68 kidneys of anesthetized rabbits. Thirty-three of the experiments were performed during or shortly after temporary renal arterial clamping. Pyelosinous backflow was observed in 67 and pyelovenous backflow in 65 of the 68 kidneys, occurring at an average intrapelvic pressure of 70 mmHg. This was true in intact kidneys and during arterial occlusion. Intrarenal backflow--intrusion of contrast material into the renal parenchyma--could be produced in only one of 35 experiments on intact kidneys, and occurred at an intrapelvic pressure of 119 mmHg. During arterial clamping, intrarenal backflow was observed in eight of nine experiments, occurring at intrapelvic pressures of about 70 mmHg. After removal of the clamp, intrarenal backflow was less frequent with shorter periods of arterial clamping and longer time between restoration of arterial flow before pyelography. Subcapsular extravasation of the medium with total blurring of the kidneys shadow and a prompt fall in intrapelvic pressure was the ultimate result of prolonged and extreme overdistension of the renal pelvis. It occurred at an average intrapelvic pressure of 80 mmHg. Histologic examination revealed tears in the fornix of the pelvic cavity in cases with pyelosinous backflow. If intrarenal backflow was present, there were tears leading from the pelvic cavity into the renal parenchyma. Supplementary experiments using a contrast material that could be demonstrated histologically (barium sulfate with gelatin) showed that the contrast filled the intertubular capillaries and venules. There was no evidence of backflow through the canalicular route.

Tissue section analysis: Feature selection and image processing

Three examples are given of the state-of-the-art of pattern recognition as regards the analysis of images of human and animal tissue: (1) the counting of cell nuclei of normal and abnormal rabbit kidney; (2) the detection of boundaries of endothelial cells of the living human cornea; and (3) the differentiation of normal and abnormal cells in the human liver. The methodology employed is that of cellular logic matched filtering using the Carnegie-Mellon SUPRPIC image processing system.

The induction of thromboxane synthetase as an expression of renal pathology

Arachidonic acid metabolism by intact perfused kidneys or by microsomes prepared from normal rabbit kidneys primarily results in the formation of prostaglandin (PG)E 2. Unilateral ureter obstruction or partial occlusion of the renal vein of rabbits leads to the release of a rabbit aorta contracting substance when such isolated perfused kidneys are stimulated with vasoactive peptides (e.g. angiotensin II and bradykinin). Homogenates of those perfused surgically modified kidneys exhibit the enzymatic conversion of either arachidonic acid or PGH 2 into thromboxane B 2. Radioimmunoassay of the renal venous effluent from the perfused ureter or renal vein obstructed kidneys demonstrated the simultaneous presence of PGE 2, thromboxane A 2, and prostacyclin in extraordinary amounts following peptide stimulation. On the other hand, the contralateral control kidney released low levels of PGE 2 and only trace amounts of thromboxane and PGI 2 when stimulated. Thus, in renal pathological states, arachidonate metabolism results in the simultaneous synthesis of a number of biologically active substances and their interplay must play an intimate role in the ultimate response of this tissue.

Atherosclerosis decreased prostacyclin formation in rabbit lungs and kidneys

Metabolism of arachidonic acid (AA) was studied in perfused lungs and kidneys of normal and atherosclerotic rabbits by determination of PGE 2, PGF 2α and the stable metabolites of PGI 2 (6-keto-PGF 1α) and TXA 2 (TXB 2). PGI 2 was the main AA metabolite formed by normal lungs and kidneys. Atherosclerosis reduced the formation of PGI 2 by about 50 % in both organs. TXA 2 formation was similarily decreased in lungs. In kidneys, the decrease in PGI 2 formation was accompanied by an increase in PGE 2 formation.

Intrarenal prostaglandins: effect of sodium and indomethacin on PGE2 and PGF2 alpha in rabbits.

The levels of prostaglandins (PGs) E2 and F2 alpha in different parts of the rabbit kidney were determined to observe the effect of sodium and indomethacin. After the pretreatment with injections of saline or with indomethacin, tissues from inner and outer medulla and cortex were separated, extracted, analyzed for PGE2 and PGF2 alpha by radioimmunoassay. In the normal rabbit kidney, the greatest amount of PG was found in the inner medulla. Saline injections appeared to increase PGE2 (but not PGF2 alpha), especially in the inner medulla. Repeated injections of saline, on the other hand, markedly reduced PGE2 in the inner medulla but increased outer medullary PGE2. Indomethacin reduced the production of PGs in all kidney segments. These results suggest the bidirectional effect of sodium on PG concentration in the rabbit kidney. Acute administration of sodium may directly stimulate the synthesis of PGE2 in the inner medulla but chronic stimulation with sodium may alter the pattern of PGE2 synthesis.

Alpha-Methyl-D-glucoside uptake in renal cortical slices of normal and alloxan diabetic rabbits.

The uptake of alpha-methyl-D-glucoside by slices of renal cortex was compared in normal and alloxan diabetic rabbits. Alloxanized rabbit tissue showed significantly higher levels of sugar accumulation than normal tissue. Diamide, which is known to oxidize intracellular glutathione (GSH), inhibited the uptake of alpha-methyl-D-glucoside by renal cortical slices. GSH stimulated sugar uptake and was also capable of reversing the inhibition of sugar accumulation caused by diamide. Mercaptoethanol and dithiothreitol, which are commonly used thiol compounds, were not as effective as GSH in stimulating sugar uptake. The level of GSH found in normal and alloxan diabetics rabbit kidneys shows a slightly decreased cortical GSH content in alloxanized animals. One can conclude that GSH participates in sugar uptake in kidney slices from both diabetic and normal rabbits.

Extraction of erythropoietin from normal kidneys.

Significant amounts of active erythropoietin were extracted from the kidneys of normal rats, cattle, dogs, and rabbits by homogenization of the organs in 0.1 M phosphate buffer. The mean erythropoietin activities of the extracts, as determined by the starved-rat assay, were 0.26 U/g beef kidney, 0.41 U/g dog kidney, and 0.11 U/g rat kidney. The dog kidney extracts had a mean activity of 0.35 U/g, as measured by stimulation of hemoglobin synthesis in cultured bone marrow cells (in vitro assay) and produced a dose-dependent stimulation of 59Fe incorporation into circulating red cells when assayed in polycythemic mice. Extracts of rabbit kidney cortices had a mean activity of 2.12 U/g, as measured by stimulation of hemoglobin synthesis in cultured bone marrow cells. When the dog kidney homogenate was fractionated on DEAE-cellulose, all of the erythropoietin activity was adsorbed to the exchanger in the presence of 0.01 M acetate buffer, pH 4.5, and was completely eluted by 0.1 M Na2HPO4-0.5 M NaCl, pH 8. An antibody made against human urinary erythropoietin completely inactivated the erythropoietic factor in the dog kidney extract. Serum from a donor dog had no erythropoietin activity when assayed in the starved rat, suggesting that the factor in the extracts is intracellular erythropoietin rather than that contained in plasma trapped in the renal vasculature. The complete inactivation of the erythropoietic factor in these kidney homogenates by antierythropoietin and its behavior on DEAE-cellulose indicate that this factor is structurally similar to native plasma erythropoietin. The extracts are completely active without being incubated in the presence of serum.

Preliminary characterization of isoimmunogenic placental antigens in the rabbit.

By isoimmunization of adult virgin female rabbits with rabbit placental extracts we have demonstrated the production of humoral antibody against two antigens, Ag-1 and Ag-2. Neither antigen is a normal serum protein, nor are they allotype proteins. Ag-1 has an alpha-2 mobility and a molecular weights of about 400,000. Ag-2 exhibits a gamma mobility but was not characterized. The antibody activity directed to Ag-1 in immune sera was the complement-fixing IgG fraction. Direct fluorescence-labeled antibody technique revealed that Ag-1 is a ubiquitous intracellular antigen primarily localized in the nucleus. The fast-growing, SV-40 virus-transformed rabbit kidney cell line (TRK-1) expressed much higher levels of Ag-1 than the slow-growing, non-transformed normal rabbit kidney cell line (LLC-RK1). The possibility that elevation of Ag-1 production is associated with rapid cell proliferation is discussed.

ANTITUBULAR BASEMENT MEMBRANE ANTIBODIES IN RENAL ALLOGRAFT REJECTION

In a patient with an episode of acute renal allograft rejection, antibodies to tubular basement membranes (TBM) were noted by direct immunofluorescence in a renal biopsy and by indirect immunofluorescence in the serum. The serum antibodies decreased gradually and became undetectable 3 months after the rejected kidney was removed. Anti-RBM antibodies eluted from the rejected kidney were capable of binding in vitro to TBM but not to glomerular basement membranes (GBM) in 39 of 40 human kidneys and various animal kidneys. The specificity was confirmed by blocking studies showing inhibition with ultrasonicated human TBM but not with GBM preparations. Passive transfer experiments showed that anti-TBM antibodies were able to bind in vivo to normal rabbit kidneys, although they could not elicit an inflammatory response. The possible mechanisms of production of anti-TBM antibodies and their potential significance in graft loss are discussed.

Thymidine Transport in Herpesvirus hominis Type 1 and 2 Infected BHK 21 Cells

Increase of dThd-uptake 4 to 12 h after infection of BHK or primary rabbit kidney cells with Herpesvirus hominis of type 1 or 2 can be considered as an early function of the virus genome, because the presence of Cyd-Ara does not prevent the increase of uptake. However, increase of uptake can be prevented by addition of actinomycin D and cycloheximide early in the synthetic cycle. Two modes of uptake have been differentiated by kinetic analysis: at low substrate concentration dThd is taken up by 'facilitated transport', whereas at high substrate concentration (above 2-5 micronM) simple diffusion takes place. The Km of transport of normal BHK or primary rabbit kidney cells (1-4 or 0-5 micronM respectively) is not changed after infection. Only the Vmax increases from 8 to 26-6 pmol in BHK cells or from 2-9 to 9-0 pmol in primary rabbit kidney cells. This indicates that 'carrier sites' with identical affinity for dThd-transport are responsible for the increase of transport after infection. This increase of transport is correlated with the induction of a virus coded thymidine kinase (TK) and not with different types of c.p.e. or cellular damage. Transport of BdUrd increases in a similar manner to that of dThd after infection; transport of dCyd or dUrd increases only slightly, whereas the mechanism of dAdo or Urd uptake by infected cells is quite different.

Relationship of cytotoxicity and interferon-inducing activity of polyriboinosinic acid. Polyribocytidylic acid to the molecular weights of the homopolymers.

SUMMARY The molecular size of poly I.poly C required for lysis of interferon-treated L cells was the same as the size required for induction of interferon in these cells. The size requirement for both interferon induction and toxicity for interferon-treated L cells resided in the poly I strand, while variations of the poly C strand size were without effect on interferon induction or cell lysis. The size of poly I.poly C required to induce interferon in primed L cells or primed primary rabbit kidney cells was the same as the size required to induce interferon in normal L cells or normal rabbit kidney cells. Again, these requirements proved more stringent for the poly I strand than for the poly C strand. These data suggest that both primed and normal cells recognize the same molecular ‘trigger’ for interferon induction.

Radioactive microsphere distribution and single glomerular blood flow in the normal rabbit kidney.

Intracortical distribution of blood flow was studied in the rabbit kidney with 15 μm labelled microspheres (M) injected into the left ventricule. M injection did not alter renal function. Thanks to arterial filling of left kidney with silicone rubber, efferent vascular patterns of the glomeruli could be precisely identified. Glomeruli of different populations were sampled by microdissection and their radioactivity measured. Assuming that intracortical distribution of M reflected distribution of flow to the glomeruli, individual glomerular blood flows (GBF) were determined. In hydropenic rabbits, GBF was higher in deep glomeruli providing vasa recta (G4) (193±14 nl·min−1) than in most other superficial (G1 and G2) and deep glomeruli (G3) (190±27, 113±10 and 127±9 nl·min−1 respectively; $$\bar m \pm SEM, n = 6)$$ . The exact significance of GBF found in superficial glomeruli with straight ascending efferent arterioles supplying aglomerular suscapsular cortex (G1) was questioned because of the possible axial streaming of spheres. Afferent medulary blood flow was calculated to represent 9.0±0.9% of total renal blood flow.

The selective inhibitory effect of hyperthermia on the metabolism and growth of malignant cells.

Hyperthermia (42° C.) exerted an inhibitory effect on the O(2) uptake of rabbit VX2 carcinoma cells in vitro, and led to a decrease in viability and growth potential of the cells, as measured by their ability to produce tumours in rabbits. Anaerobic glycolysis of the tumour cells was unaltered by hyperthermia. Respiration and anaerobic glycolysis of normal rabbit liver, kidney and red blood cells were unaffected by the elevated temperature. Local heat was applied to established VX2 tumours in vivo, with a subsequent reduction in tumour size in all cases, the most effective therapy regime being 3 one-hour applications of heat within the mean cell generation time of the tumour. Following heating there was rapid and widespread tumour cell necrosis and lysis, with subsequent replacement of the tumour architecture by connective tissue. There was a prolongation of survival time in 50% of the treated animals, which are still alive 18 months after therapy; all the control animals died within 10 weeks. The selective inhibitory effects of hyperthermia on cancer cells, and its application to human neoplasms, are discussed.

Inactivation of erythropoietin by tissue homogenates.

Homogenates prepared from normal rabbit kidneys were found to contain a strong inhibitor of human erythropoietin obtained from human urine or from human plasma. The kinetics of the inactivation suggests it to be a nonenzymatic process. Dialysis against water or EDTA saline did not change the activity of the inhibitor and heating to 100°C did not completely abolish its activity. The addition of normal plasma reduced the inhibitory activity of the renal homogenate, a reduction that was directly proportional to the amount of normal plasma added and was observed both with urinary erythropoietin and with plasma erythropoietin. Homogenates prepared from normal rabbit spleens and livers were also found to contain an inhibitory principle that was modified by the addition of normal plasma. Homogenates from normal rabbit bone marrow were almost devoid of inhibitory activity.

Some biological characters of cell lines derived from normal rabbit kidney.

Some biological characters of cell lines derived from normal rabbit kidney.

Acute Vascular Lesions Produced by Selected Non-pressor Rental Cortical Extracts

Pressor and non-pressor aqueous extracts were separated from normal and ischemic rabbit kidney cortex by column chromatography and injected into test animals. Five to 6 test animals injected with the non-pressor extract from ischemic kidney developed vascular injuries especially in small cerebral arteries. This finding suggests that a non-pressor substance capable of producing vascular injury independent of renin is present in ischemic kidney cortex.

STUDIES ON THE SERUM AND THE INFECTIOUS HEPATITIS VIRUSES OF MAN. II. COMPLEMENT FIXATION STUDIES WITH SERUM AND INFECTIOUS HEPATITIS VIRUSES.

Serum hepatitis (SH) virus was isolated in tissue culture from the blood of each of the five human volunteers 41 to 90 days following their inoculation with NIH icterogenic plasma pool VI. Six other strains of hepatitis viruses were isolated from patient sources; one from a case of clinical serum hepatitis and the remainder from five (5) cases of clinically diagnosed infectious hepatitis. All agents were grown in human lung cell and rabbit kidney tissue culture systems. SH 204 virus was neutralized by specific rabbit antisera prepared against viruses SH 211, SH 204, SH 210, SH‐Neib, IH‐MJ and IH‐Horst. These antisera at a final dilution of 1:40 neutralized 10 to 1,000 TCID50 units of the SH 204 virus. Cross complement fixation tests were carried out with the ten hepatitis viruses against specific rabbit antisera which was prepared against each of the virus agents. Irrespective of the source of isolation of the SH or IH virus, there were no apparent differences in the antigenic structure of the viruses, since all produced a common complement fixing antibody in rabbits immunized with the different viral agents. The complement fixation titers observed ranged from 1:200 to 1:3000. In sharp contrast, normal human serum and normal rabbit kidney and human lung cell tissue culture fluids, when tested against antisera produced to these agents as antigens, fixed complement in a range of titer of only 1:8 to 1:96.Complement fixation tests were carried out against specific antisera for many of the other human enteric viruses. SH virus did not fix complement with specific antisera prepared against Coxsackie, Group B1 virus types one to six; Coxsackie, Group A, Virus, type nine; Echo, types one, three, six, eight, 23, 26, 28; Folio viruses, types one, two and three; and Para‐influenza, type three virus. However, the SH virus was found to fix complement with antisera prepared against Echo virus, types two, seven, 24 and 25, but the titers observed ranged only from 1:64 to 1:128. Complement fixing antibodies were also demonstrated in three of 25 normal calf sera tested, but were not demonstrated in normal serum obtained from guinea pigs, rabbits, rhesus monkey, horse, human infant, sheep or cat. Antisera against infectious canine hepatitis virus did not fix complement with the SH virus. Finally, convalescent sera obtained from the volunteers who had been inoculated with the icterogenic plasma pool, demonstrated a high titer of complement fixing antibodies against the SH virus.