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Biomedical subjects

S Nielsen

Publications and source records attributed to S Nielsen.

At least 271 records · Page 15Linked to original sources
PubMed1993

Stepwise degradation of NT in pars convoluta and recta of rabbit proximal tubules: evidence of axial heterogeneity.

Reabsorption and degradation of the neuropeptide neurotensin (NT) in rabbit proximal pars convoluta (PC) and pars recta (PR) nephron segments were characterized. Brush-border membrane vesicle fractions (PC or PR) were incubated with [3H]NT, and the extent and pattern of peptide hydrolysis were determined by reversed-phase high-pressure liquid chromatography (rHPLC). Furthermore, isolated rabbit PC and PR segments were perfused with [3H]NT, reabsorption of [3H]NT was quantified, and the collected perfusate was analyzed by HPLC. Metabolites were characterized. Finally, rabbit proximal tubules were microinfused in vivo with [3H]NT to follow the tubular uptake by electron microscope autoradiography. Degradation increased with time in both vesicle fractions. The main difference was an extensive cleavage of NT in PR, as revealed by a higher proportion of end metabolites. This was also visualized as a higher proportion of the large degradation product in rHPLC fraction 39 [NT-(1-11)] in PC as compared with PR after 30 min of incubation. The isolated perfused proximal tubular segments processed NT with large efficiency. PC segments processed 90% of the perfused amount, and PR processed 88%. Only 13% in PC and 10% in PR of the processed NT were found in the bath and the tubule. The main part of processed NT was in the collected perfusate, and rHLPC profiles revealed that NT-(1-11) was the only metabolite in both PC and PR. Electron microscope autoradiography demonstrated autoradiographic grains over invaginations and over the apical part of the proximal tubule cell in endocytic vesicles and vacuoles 10 min after microinfusion of [3H]NT.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1993

Kinetics of endocytosis in renal proximal tubule studied with ruthenium red as membrane marker.

This study was performed to determine the membrane area of the different compartments involved in endocytosis in rat kidney proximal tubule. This enables a direct estimation of the kinetics of the membrane turnover in the subprocesses of endocytosis and recycling of membrane constituents. To accomplish this, cross-sectioned invaginations have to be distinguished from endocytic vesicles. To obtain a continuous heavy staining of the apical plasma membrane, we have developed a sandwich-staining technique involving initial staining with ruthenium red, osmium, and thiocarbohydrazide functioning as a molecular bridge between osmium molecules. The distribution of invaginations, noncoated and coated endocytic vesicles, as well as the surface density of invaginations, small endocytic vesicles, large endocytic vacuoles, lysosomes, and dense apical tubules are determined in segment 1 (S1) and 2 (S2) of the proximal tubule. This has shown that invaginations constitute most [54% (S1 and 62% (S2)] of the membrane in small membrane-bounded structures in the apical cytoplasm. No morphological characteristics enabled direct differentiation between cross-sectioned invaginations and small endocytic vesicles, but a method for correct identification of 72% of all invaginations and 82% of all small endocytic vesicles is presented. Using the surface densities of various compartments together with previous data on the internalization of membrane markers, we developed a kinetic model enabling calculation of the velocity of membrane internalization and subsequent recycling. The velocity of internalization is 6.4 X 10(-3) microns2.micron-3.s-1, corresponding to internalization of a membrane area equivalent to the entire surface of invaginations within 78 s. The velocity of membrane transfer from the vacuolar compartment to dense apical tubules and of recycling from dense apical tubules to the luminal membrane is 6.2 x 10(-3) microns 2.microns-3.s-1. The entire membrane area equivalent to the surface of the vacuolar compartment and dense apical tubules is transferred in 43 and 92 s, respectively. The amount of membrane that surrounds lysosomes is transported from the vacuolar compartment within 23 min.

Animals↗
PubMed1993

Vasopressin activates collecting duct urea transporters and water channels by distinct physical processes.

The present studies were performed to investigate the kinetics of regulation of water channels and urea carriers in the rat terminal (IMCD) in response to vasopressin (AVP). The time courses of osmotic water permeability (Pf) and urea permeability (P(urea)) were measured in isolated perfused rat terminal IMCD segments following AVP stimulation and subsequently following AVP washout. Under control conditions, Pf and P(urea) kinetics were similar. Both transport processes exhibited complex patterns of activation with a period of rapid permeability increase followed by a period of slower increase. Both transporters also exhibited complex patterns of reversal following AVP washout, with a rapid permeability decrease (5 min) followed by a slower decrease toward the baseline value. The measurements were repeated in the presence of a lumen > bath osmotic gradient, a condition associated with a decreased rate of apical endocytosis in collecting ducts. The lumen > bath gradient did not alter the kinetics of Pf increase after AVP addition, but completely blocked the decrease in Pf normally seen with washout of AVP. In contrast, the lumen > bath osmotic gradient did not affect the decrease in urea permeability after AVP washout, but blocked the rapid phase of urea permeability increase following AVP addition. Thus imposition of a lumen > bath osmotic gradient resulted in separation of the time courses of P(urea) and Pf changes associated with AVP addition and washout. This finding indicates that the physical processes responsible for AVP-mediated alteration of urea transporter and water channel activity in the apical membrane are distinct.

Animals↗
PubMed1993

Kinetic model of water and urea permeability regulation by vasopressin in collecting duct.

We present a mathematical model describing the kinetics of water-channel and urea-carrier regulation by vasopressin in the apical membrane of collecting duct cells. The rate of change of the number of activated channels or carriers in the apical membrane is modeled as a balance between the rate of activation (or exocytic insertion) and the rate of inactivation (or endocytic retrieval) of transporters. In a three-state version of the model, transporters are assumed to be partitioned into three physical states, i.e., an "activated" state that imparts a permeation pathway to the apical membrane, an "inactivated" state, and a "reserve" state. Both activation and inactivation are represented by first-order kinetic equations describing transition from reserve to activated transporters and from activated to inactivated transporters, respectively. A simplified two-state model is derived from the three-state model, with the assumption that the transformation from inactivated to reserve transporter occurs rapidly relative to the other state transitions. Simulated time courses obtained by solving model equations are compared with experimentally determined time courses to test whether the response to vasopressin in isolated inner medullary collecting duct segments can be explained by direct effects on the rate constants for activation or inactivation. The results indicate that, for both transporters, it must be assumed that vasopressin directly regulates rate constants for both activation (exocytosis) and inactivation (endocytosis) to account for the experimentally determined dynamic responses to vasopressin and its withdrawal. These studies provide a theoretical basis on which to design further experimental studies.

Animals↗
PubMed1993

Vasopressin- and cAMP-induced changes in ultrastructure of isolated perfused inner medullary collecting ducts.

Studies were performed to correlate arginine vasopressin (AVP)-induced changes in epithelial ultrastructure with changes in osmotic water permeability in isolated perfused rat terminal inner medullary collecting ducts (tIMCD). The tubules were perfused in three time periods, i.e., a 40-min basal period, a 40-min period with 0.1 nM AVP in the bath, and a 60-min withdrawal period. In each phase, the osmotic water permeability (Pf) was measured, and the perfused tubules were fixed for electron microscopy. AVP caused a four- to eightfold increase in Pf and induced several ultrastructural changes as follows: increased cell height of IMCD cells, expansion of the intercellular spaces, formation of large vacuoles, and increased coated pit density in the apical plasma membrane [from 0.6 +/- 0.2 (n = 6) to 2.9 +/- 0.3 (n = 7) pits/100 microns membrane length]. During AVP withdrawal, Pf decreased toward the basal value in association with partial reversal of the ultrastructural changes including a decrease in coated pit density to 1.0 +/- 0.2 (n = 4). Stimulation with 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) (0.1 mM) produced similar changes in Pf. Coated pit density increased to 2.1 +/- 0.4 (n = 4) after cAMP stimulation and after cAMP withdrawal decreased to 1.2 +/- 0.2 (n = 6). In contrast to stimulation with AVP, cAMP stimulation did not result in dilated intercellular spaces or formation of large vacuoles. The only ultrastructural feature that directly correlated with the water permeability was the density of coated pits in the apical membrane. Organelles involved in the endocytic pathway were studied with cationized ferritin or albumin-gold in the luminal perfusate. At the end of 40 min basal perfusion or AVP stimulation, luminal tracer was found almost exclusively in large multivesicular bodies (MVB). Tubules perfused with tracer during AVP withdrawal demonstrated rapid tracer accumulation in small vesicles and small MVB within 3-5 min, a time point corresponding to the rapid phase of Pf decrease. Later (30-60 min) the label was mainly confined to large MVB. Occasionally during AVP stimulation or withdrawal, small coated vesicles and smooth vesicles with coated extensions were noted to contain tracer. The data demonstrate AVP-mediated coated pit formation and cellular changes and show very rapid internalization of apical membrane after AVP withdrawal.

Animals↗
PubMed1993

Aquaporin CHIP: the archetypal molecular water channel.

Despite longstanding interest by nephrologists and physiologists, the molecular identities of membrane water channels remained elusive until recognition of CHIP, a 28-kDa channel-forming integral membrane protein from human red blood cells originally referred to as "CHIP28." CHIP functions as an osmotically driven, water-selective pore; 1) expression of CHIP conferred Xenopus oocytes with markedly increased osmotic water permeability but did not allow transmembrane passage of ions or other small molecules; 2) reconstitution of highly purified CHIP into proteoliposomes permitted determination of the unit water permeability, i.e., 3.9 x 10(9) water molecules.channel subunit-1 x s-1. Although CHIP exists as a homotetramer in the native red blood cell membrane, site-directed mutagenesis studies suggested that each subunit contains an individually functional pore that may be reversibly occluded by mercurial inhibitors reacting with cysteine-189. CHIP is a major component of both apical and basolateral membranes of water-permeable segments of the nephron, where it facilitates transcellular water flow during reabsorption of glomerular filtrate. CHIP is also abundant in certain other absorptive or secretory epithelia, including choroid plexus, ciliary body of the eye, hepatobiliary ductules, gall bladder, and capillary endothelia. Distinct patterns of CHIP expression occur at these sites during fetal development and maturity. Similar proteins from other mammalian tissues and plants were later shown to transport water, and the group is now referred to as the "aquaporins." Recognition of CHIP has provided molecular insight into the biological phenomenon of osmotic water movement, and it is hoped that pharmacological modulation of CHIP function may provide novel treatments of renal failure and other clinical problems.

Amino Acid Sequence↗
PubMed1993

Luminal and basolateral uptake and receptor binding of IGF-I in rabbit renal proximal tubules.

The aim of the present study was to quantify and compare the luminal and basolateral binding and uptake of 125I-labeled insulin-like growth factor I (IGF-I) by means of 1) isolated, perfused, proximal tubules combined with electron microscope autoradiography and 2) luminal and basolateral membrane vesicles from rabbit proximal tubules. 125I-IGF-I was added to isolated perfused proximal tubules for 30 min in concentrations of 1.6-3.9 micrograms/l to either the perfusate or the bath. The luminal and basolateral uptake in 30 min averaged 447 and 410 fg/mm, respectively. About 20% of the luminally absorbed IGF-I was digested. Addition of excess unlabeled IGF-I (10(-7) M) to the bath produced complete inhibition of the basolateral binding/uptake, whereas no inhibition of the luminal uptake was seen. Electron microscope autoradiography showed that IGF-I after luminal endocytic uptake to a large extent was transported into lysosomes. After basolateral exposure the major portion of the grains was found over the basolateral cell membrane; however, a significant amount was located over endocytic vacuoles and lysosomes in both apical and basal parts of the cells. In both luminal and basolateral membrane vesicles, single-class, high-affinity binding sites for IGF-I were found with dissociation constants of 6.3 and 5.7 nM, respectively. Specific binding capacities averaged 2.7 and 25.7 pmol IGF-I/mg protein in luminal and basolateral vesicles. The biochemical data suggest an asymmetric distribution of specific IGF-I receptors in the luminal and basolateral membranes, with a greater abundance of receptors in the latter. The extensive basolateral endocytic binding/uptake of IGF-I compared with that of the luminal in isolated perfused tubules differs considerably from the processing of other peptide hormones.

Animals↗
PubMed1993

Structural-functional correlation in chinchilla long loop of Henle thin limbs: a novel papillary subsegment.

The ultrastructural characteristics of thin limb subsegments from chinchilla long loops of Henle were studied in perfusion-fixed kidneys and in isolated perfused tubules. In sections from the perfusion-fixed kidneys, we noted types I, II, III, and IV thin limb epithelia similar to those previously identified in other rodent species. Sections from the deepest 20% of the papillary tip, however, revealed only a single thin limb epithelial type, which had a combination of structural characteristics distinct from previously identified thin limb subtypes. This "papillary type" epithelium had relatively tall cells and a complex cellular organization with extensive interdigitation, numerous shallow tight junctions, and microvilli. In single-tubule studies, thin limb segments dissected from different levels of the outer and inner medulla were perfused in vitro for osmotic water permeability (Pf) measurements and were fixed for ultrastructural examination. Long-loop thin descending limbs (LDL) dissected from the outer medulla (Pf, 2,637 +/- 336 micron/s) had type II epithelium. LDL dissected from the middle of the inner medulla (Pf, 1,570 +/- 76 microns/s) had a type III epithelium. LDL segments dissected from the deepest 20% of the inner medulla had a low but nonzero Pf (68 +/- 9 micron/s) and had the same novel papillary type epithelium seen in sections from fixed kidneys. Thin ascending limbs dissected from inner 50% of the inner medulla had essentially zero Pf (8 +/- 4 micron/s) and had a type IV epithelium. Immunohistochemical localization of CHIP28 water channel protein confirmed the presence of CHIP28 in thin descending limbs throughout the outer 75% of the inner medulla, whereas labeling was essentially absent in the deep inner medulla where the low-PfLDL (novel papillary type epithelium) is located.

Animals↗
PubMed1993

Concurrent expression of erythroid and renal aquaporin CHIP and appearance of water channel activity in perinatal rats.

Major phenotypic changes occur in red cell membranes during the perinatal period, but the underlying molecular explanations remain poorly defined. Aquaporin CHIP, the major erythroid and renal water channel, was studied in perinatal rats using affinity-purified anti-CHIP IgG for immunoblotting, flow cytometry, and immunofluorescence microscopy. CHIP was not detected in prenatal red cells but was first identified in circulating red cells on the third postnatal day. Most circulating red cells were positive for CHIP by the seventh postnatal day, and this proportion rose to nearly 100% by the 14th day. The ontogeny of red cell CHIP correlated directly with acquisition of osmotic water permeability and inversely with Arrhenius activation energy. Only minor alterations in the composition of red cell membrane lipids occurred at this time. Immunohistochemical analysis of perinatal kidneys demonstrated a major induction of CHIP in renal proximal tubules and descending thin limbs at birth, coincident with the development of renal concentration mechanisms. Therefore, water channels are unnecessary for oxygen delivery or survival in the prenatal circulation, however CHIP may confer red cells with the ability to rehydrate rapidly after traversing the renal medulla, which becomes hypertonic after birth.

Aging↗
PubMed1993

Systolic blood pressure relates to the rate of decline of glomerular filtration rate in type II diabetes.

OBJECTIVE: To relate deterioration in kidney function to some potential cardiovascular risk factors in type II diabetic patients. RESEARCH DESIGN AND METHODS: Twenty-four normoalbuminuric and 13 microalbuminuric patients completed a 3.4-yr prospective observational study. Glomerular filtration rate, urinary albumin excretion rate, blood pressure, glycemic control, and lipids were measured on entry and at the end of the study. Of the patients, 19 normoalbuminuric and 8 microalbuminuric (73%) patients had no history of antihypertensive treatment. RESULTS: The glomerular filtration rate was significantly reduced during the follow-up period (-1.34 +/- 0.54 ml.min-1.1.73 m-2 [mean +/- SE], P < 0.02). The rate of decline varied considerably in normoalbuminuric and microalbuminuric patients (from -13.5 to 4.3 and from -7.0 to 4.2 ml.min-1.1.73 m-2 per year, respectively) but was on average not accelerated in normoalbuminuric or microalbuminuric patients (-1.3 +/- 0.7 and -1.5 +/- 0.8 ml.min-1.1.73 m-2 per year, respectively). Significant correlations were observed between the glomerular filtration rate fall rate and initial systolic blood pressure (r = -0.47, P < 0.01; patients without antihypertensive treatment: r = -0.42, P = 0.03) but not diastolic blood pressure. In a stepwise multiple linear regression analysis, baseline systolic blood pressure significantly determined the fall rate of the glomerular filtration rate (regression coefficient = -0.050, SE = 0.018, P = 0.011; patients without antihypertensive treatment: regression coefficient = -0.047, SE = 0.021, P = 0.030). CONCLUSIONS: In these type II diabetic patients neither normoalbuminuria nor microalbuminuria are at an average associated with an accelerated decline in kidney function. Still, systolic blood pressure is a determining factor for the rate of decline in the glomerular filtration rate. A longer follow-up time with consecutive glomerular filtration rate measurements are needed to determine the long-term implications of normoalbuminuria and microalbuminuria on kidney function in type II diabetic patients.

Aged↗
PubMed1992

Novel functional M1 selective muscarinic agonists. Synthesis and structure-activity relationships of 3-(1,2,5-thiadiazolyl)-1,2,5,6-tetrahydro-1-methylpyridines .

A series of novel 3-(3-substituted-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro- 1-methylpyridines (substituted-TZTP; 5a-l, 7a-h, 8, 9c-n, 11, 13j) were synthesized and tested for central muscarinic cholinergic receptor affinity by using [3H]-oxotremorine-M (Oxo-M) and [3H]-pirenzepine (Pz) as ligands. The potency and efficacy of the compounds for the pharmacological defined M1 and M2 muscarinic receptors were determined on isolated electrically stimulated rabbit vas deferens and on spontaneously beating isolated guinea pig atria, respectively. Selected compounds were also tested for M3 activity in the isolated guinea pig ileum. The C1-8 alkoxy-TZTP 5a-l analogues all displaced [3H]-Oxo-M and [3H]-Pz with low nanomolar affinity. Depicting chain length against Oxo-M binding and against Pz binding the unbranched C1-8 alkoxy-TZTP (5a-h) derivatives produced U-shaped curves with butoxy- (5d) and (pentyloxy)-TZTP (5e) as the optimum chain length, respectively. This U-shaped curve was also seen in the ability of the compounds 5a-h to inhibit the twitch height in the vas deferens preparation. The (pentyloxy)- (5e) and the (hexyloxy)-TZTP (5f) analogues produced an over 90% inhibition of the twitch height with IC50 values in the low picomolar range. In both the atria and in the ileum preparations 5f had low efficacy and potency. With the (alkylthio)-TZTP (7a-h) analogues the structure-activity relationship was similar to the one observed with the alkoxy (5a-h) analogues, but generally 7a-h had higher receptor affinity and was more potent than the corresponding 5a-h. However, the C3-8 alkyl-TZTP (9c,e,g,h) analogues had 10-100 times lower affinity for the central muscarinic receptors than the corresponding alkoxy and alkylthio derivatives, and their efficacy in the vas deferens preparation was too low to obtain IC50 values. The unsubstituted TZTP (11) compound was a potent but nonselective muscarinic agonist. The two 3-(3-butoxy/(hexyloxy)-1,2,5-oxadiazol-4-yl)-1,2,5,6-tetrahy dro-1- methylpyridines (butoxy/hexyloxy)-OZTP; 19a/b) were also synthesized and tested. Both 19a and 19b had much lower affinity for the central muscarinic receptors than 5d and 5f, and the efficacy of 19a,b was too low to give IC50 values in the vas deferens preparation. Therefore, the C5-6 (alkyloxy)/(alkylthio)-TZTP's represent a unique series of potent functional M1 selective muscarinic agonists.

Animals↗
PubMed1992

Time course and kinetics of proximal tubular processing of insulin.

The present study was undertaken to determine the time courses and kinetics of the subcellular processing of 125I-insulin in isolated and in vitro perfused proximal tubules. Morphometric analysis demonstrated well-preserved ultrastructure after 90 min of perfusion. After luminal perfusion for 90 min the absorption was constant with time and reached steady state within 5 min (177 +/- 7 fg.min-1.mm-1). Also the hydrolysis rate and tubular accumulation rate were constant and averaged 84 +/- 8 and 93 +/- 10 fg.min-1.mm-1, respectively. Free 125I appeared already within 5 min of perfusion and reached steady state within 10 min. From proximal tubules perfused with 125I-insulin for 30 min and chased for 60 min, a compartmental analysis revealed two compartments; half time (t1/2) for delivery of insulin to the lysosomes was determined to be 8.5 min, and t1/2 for lysosomal degradation was 72 min. The results demonstrated that internalization by endocytic invaginations, incorporation in endocytic vacuoles, fusion with lysosomes, and hydrolysis were rapid processes and reached maximum rates within few minutes. A significant transtubular transport of insulin to the peritubular compartment was determined to be a constant rate of 11.2 +/- 0.7 fg.min-1.mm-1. Perfusion of tubules with insulin at high concentrations in the perfusate revealed that the transport was dependent on the absorbed amount and not on the perfused load, compatible with transport through the cells and not via a paracellular mechanism. The intactness of the tight junctions was supported by the following: 1) [14C]inulin leak did not increase with time and 2) enzyme-free intercellular spaces were evident after perfusion for only 5 min with microperoxidase (mol wt of 1,700). The transported 125I-insulin was trichloroacetic acid precipitable and immunoprecipitable.

Animals↗
PubMed1992

The press-fit Kinemax knee arthroplasty. High failure rate of non-cemented implants.

We report the results of 75 consecutive primary press-fit Kinemax arthroplasties with an average follow-up of 14 months (three to 28). We reviewed 26 cemented and 49 non-cemented tibial components implanted into 72 patients (30 men and 42 women, median age 70 years). At the latest follow-up the overall evaluation (Hospital for Special Surgery knee rating scale) for cemented cases was excellent in 54%, good in 42% and poor in 4%. No cemented prosthesis loosened. Of the non-cemented cases 55% were excellent, 23% good, and 2% fair. Ten tibial implants (20%) loosened and required revision. Residual pain marred the result in 24% of the non-cemented prostheses and in 4% of the cemented group. We do not recommend the press-fit, smooth-surfaced Kinemax prosthesis for non-cemented use.

Adult↗
PubMed1992

Microalbuminuria in non-insulin-dependent diabetes.

According to international consensus, microalbuminuria is defined as an elevated urinary albumin excretion rate (UAER) of 20-200 micrograms/min, which is below the proteinuric range. Nephropathy is a major complication in IDDM, seen in about 30% of patients after many years of diabetes. Increasing microalbuminuria is an excellent marker of subsequent nephropathy in these patients. End-stage diabetic nephropathy is also important in NIDDM, but in most Western countries this serious complication eventually develops in only 5 to 10% of cases, whereas the majority of patients die before this from cardiovascular disease. In completely healthy individuals there is no clear correlation between age and UAER, at least up to about 70 years of age. The mean excretion rate is around 5 micrograms/min, with a considerable range, but excretion only rarely exceeds 15 micrograms/min. In population studies among middle-aged and elderly individuals, higher values are seen. In newly diagnosed NIDDM about 40% of patients show an excretion rate above 15-20 micrograms/min. There is a significant but not precise correlation between albumin excretion rate and glycemic control, and usually UAER is reduced by standard antidiabetic treatment. In a considerable number of patients, high values cannot be reduced. In the course of NIDDM about 20-30% of patients show microalbuminuria. In patients with known diabetes, microalbuminuria is related not only to subsequent diabetic proteinuria, but even more strongly to early death, mainly from cardiovascular disease. Even slight microalbuminuria (15-40 mg/l in early morning urines) is clearly associated with increased mortality. In subjects with newly detected elevated blood glucose (by screening) microalbuminuria also predicts early mortality. The mechanisms are not established, but several arteriosclerosis-related risk factors are seen more frequently in patients with microalbuminuria, e.g. lipid abnormalities, elevated systolic blood pressure (BP), hemostatic measures, as well other markers of cardiovascular disease. Usually there is a significant but not precise correlation between BP and UAER in groups of patients throughout the course of diabetes. New studies document that also in the elderly background population microalbuminuria is a significant risk factor for early death, maybe even stronger than the established risk markers, which thus may be confounded with the presence of microalbuminuria.

Adult↗