Multi-site, multi-country evaluation of analytical and operational performance of a low-mid volume chemiluminescent immunoassay analyzer.

BACKGROUND: A new automated immunoassay low-mid volume (< or = 250 immunoassays/day) chemiluminescent analyzer, Abbott Architect i1000sR, was evaluated by seven laboratories around the world (4 in Europe, one each in Canada, Japan, and the U.S.A.) to demonstrate equivalent performance for key operating characteristics (e.g., precision, turn around time, limit of detection, functional sensitivity, and linearity). METHODS: The laboratories followed standard protocols to assess precision, limit of detection (LoD), functional sensitivity, assay linearity, method comparison, and sample carryover. Turn around time for three stat assays (beta-hCG, BNP, and CK-MB) and the time required to complete workloads of 50 and 100 tests with a mixture of 75% routine tests and 25% stat tests was also evaluated. RESULTS: Total precision was typically < 5% CV for nine immunoassays. Analytical performance met design goals and demonstrated equivalency to package insert data for assays on market and in use for an existing high volume immunoassay system. Stat turn around times were consistent with the fixed analytical time of 15.6 minutes and met the expectations of the laboratories. Measured test throughput ranged from 47 - 54 tests per hour and demonstrated that the analyzer was fit for the intended purpose of supporting a laboratory that performs < or = 250 immunoassays per day. CONCLUSIONS: A multisite, international analyzer familiarization study is a practical means of confirming that a new instrument meets both a manufacturer's design specifications and users' real world expectations and provides a pragmatic test for the system. The experience of investigators at seven sites around the world indicates that a new fully automated chemiluminescent system is suitable for use.

Pathways for organic osmolyte synthesis in rabbit renal papillary tissue, a metabolic study using 13C-labeled substrates.

Renal papillary collecting duct cells have been postulated to adapt their intracellular osmolality to the large changes in interstitial osmolality by changing their content of 'non-perturbing' organic osmolytes such as sorbitol and myo-inositol. 13C-NMR was used in this study to elucidate the metabolic pathways leading to a synthesis of those compounds. Incubation of rabbit renal papillary tissue with [1-13C]glucose showed label scrambling mainly into sorbitol (C-1) and lactate (C-3). This result confirms activity of aldose reductase and glycolytic enzymes in renal papillary cells. Using [3-13C]alanine or [2-13C]pyruvate as carbon source, 13C-labeling of sorbitol and myo-inositol was observed, indicating that renal papillary tissue possesses, in addition, gluconeogenic activity. The latter assumption is supported by the result that in enzyme assays rabbit kidney papilla and isolated rat kidney papillary collecting duct cells show significant fructose-1,6-bisphosphatase activity.

Control of sorbitol metabolism in renal inner medulla of diabetic rats: regulation by substrate, cosubstrate and products of the aldose reductase reaction.

Streptozotocin diabetes induces a 4-fold increase in the maximal velocity of inner medullary aldose reductase as determined in vitro but increases sorbitol synthesis in intact inner medullary collecting duct (IMCD) cells only 1.3-fold. In order to resolve this discrepancy we investigated the importance of intracellular factors in controlling the role of cellular sorbitol synthesis. These factors include glucose concentration, sorbitol concentration, the activity of the NADPH-regenerating pentose phosphate pathway, intracellular NADP and NADPH content, and intracellular reduced (GSH) and oxidized glutathione (GSSG). It was found that the apparent Km of cellular sorbitol production for glucose was identical in control and diabetic rats (56 +/- 18 vs. 59 +/- 14 mmol/l D-glucose), whereas Vmax increased by 31% in diabetes. In inner medullary collecting duct cells of diabetic rats containing 146 +/- 5 mumol sorbitol/g protein, sorbitol synthesis was slightly lower (-15%), compared to cells which had been sorbitol-depleted prior to the experiment (87 +/- 4 mumol sorbitol/g protein). However, no inhibitory effect of sorbitol (up to 200 mmol/l) was observed on aldose reductase activity in vitro. In diabetic rats the content of NADPH was about 32% lower than in the control rats (3.8 +/- 0.3 vs. 5.6 +/- 0.4 mumol/g protein) and the ratio of NADPH/NADP was decreased from 25.6 +/- 5.1 to 8.6 +/- 1.7. In homogenates of the inner medulla the activity of 6-phospho-gluconate dehydrogenase (EC 1.1.1.43) was identical in both experimental groups, so the pentose phosphate shunt seems to be unaltered. GSH content in diabetic rats was also diminished (4.02 +/- 0.67 mumol/g protein vs. 7.41 +/- 0.5 mumol/g protein) and the GSH/GSSG ratio fell from 92.6 to 57.4. In enzyme tests in vitro an apparent Km of 7.3 +/- 1.9 mumol/l of the aldose reductase for NADPH was found; NADP acted as competitive inhibitor with an apparent K(i) of 183 +/- 31 mumol/l. Aldose reductase activity was also found to be strongly inhibited by the SH-group reagent p-chloromercurybenzoesulfonate (apparent K(i) = 0.85 x 10(-6) mol/l). Combining the results obtained on the properties of the aldose reductase in vitro and the observation made in the intact cells, the investigators suggest that the decrease in NADPH/NADP ratio, as well as changes in the redox state in the cells of diabetic animals, can play a significant role in the control of sorbitol synthesis.

Pleiotropic effects of hepatocyte growth factor in proximal tubule involve different signaling pathways.

Hepatocyte growth factor (HGF) accelerates renal tubule cell regeneration and induces tubulogenic differentiation via the intracellular tyrosine kinase (TK) domain of its receptor, the proto-oncogene c-Met. We tested whether different signaling pathways may be involved by examining HGF binding and effects on cell proliferation, migration, scattering, and tubulogenic differentiation in the bipolar differentiating rabbit proximal tubule cell line PT-1 under serum-free conditions in the presence or absence of the protein TK inhibitors (PTKIs) herbimycin-A, genistein, methyl-2,5-dihydroxycinnamate, and geldanamycin. These PTKIs inhibit pp60(c-src), a nonreceptor TK involved in cell-growth control. HGF bound to a single high-affinity receptor class, increased microvilli numbers 1.5-fold, enhanced cell proliferation and migration 1.8-fold, and stimulated formation of tubule structures 2.2-fold. PTKI inhibited the mitogenic and motogenic effects of HGF with different potencies and comparable maximal effects but had no specific influence on HGF-induced tubulogenic cell differentiation. These data underline the importance of pp60(c-src) in mediating mitogenic and motogenic effects of HGF, whereas stimulation of tubulogenic cell differentiation may be transduced by a pp60(c-src)-independent pathway.

Different binding and degradation of proinsulin, insulin and insulin-like growth factor-1 (IGF-1) in cultured renal proximal tubular cells. Implications for the prolonged serum half-life of proinsulin.

Recent studies have reported that elevated proinsulin levels are indicative of an increased cardiovascular risk. Renal proximal tubular cells represent a major site for the metabolism of insulin-like hormones after glomerular filtration into the tubular lumen. To determine the binding and degradation of proinsulin in comparison with insulin and insulin-like growth factor-1 (IGF-1), we have used a rabbit proximal tubular cell line (PT-1). As confirmed by electron microscopy. PT-1 cells exhibit bipolar differentiation, demonstrating apical microvilli and invaginations of the basolateral membrane. To allow selective incubation of both compartments, cells were grown on filter membranes. Performing equilibrium binding assays with 125I-labelled hormones, severalfold higher binding was found at the apical than at the basolateral cell membrane, with the capacity range IGF-1 > insulin > proinsulin. Half-maximal displacement of 125I-labelled insulin and IGF-1 was observed at 0.6 and 2 nM, respectively, while crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Half-maximal displacement of 125I-proinsulin binding was obtained at approx. 8 nM proinsulin and insulin, whereas IGF-1 was 10-fold less potent. The relative degradation of specifically bound tracer was lowest for proinsulin (apical 10%, basolateral: 13%). IGF-1 was degraded by 20% at the apical cell membrane, and up to 78% at the basolateral membrane. In contrast, almost the total amount of insulin bound was degraded at both membrane sites (apical 99%, basolateral: 83%). These results suggest separate insulin and IGF-1 receptors while proinsulin binds with high affinity to a third insulin-like receptor on the apical membrane of PT-1 cells.

Blood rheology after LDL apheresis using dextran sulfate cellulose absorption--a case report.

The authors describe a thirty-eight-year-old woman with familial hypercholesterolemia treated by dextran sulfate cellulose adsorption apheresis. This technique and the selective extracorporeal LDL cholesterol elimination by immunoabsorption or heparin-induced precipitation not only dramatically decrease blood lipids but also result in a marked improvement in the rheologic profile. It is suggested that the amelioration of blood rheology by LDL apheresis may represent the cause for the early clinical improvement felt by most patients with severe coronary heart disease and hypercholesterolemia.

Clearance of osteocalcin in adults with end-stage renal disease undergoing CAPD.

Osteocalcin (OC) is a bone-specific protein whose blood concentration is a specific and sensitive marker of bone turnover. In adults undergoing continuous ambulatory peritoneal dialysis (CAPD), mean serum osteocalcin levels (S-OC) are lower than in similar patients on hemodialysis. We therefore measured the serum (S) and dialysate (D) levels of OC, estimated the peritoneal clearance (Cp) and mass transfer (MT) of OC and evaluated the relationship between S-OC levels and other serum biochemical parameters of bone metabolisms. Fourteen adult patients on CAPD were studied with a mean age of 46.3 +/- 13 years and a mean dialytic age on CAPD of 17.4 +/- 9.6 months. OC concentrations in (S) and (D) were 60.8 +/- 55.5 micrograms/l (normal range: 4.3-12.4 micrograms/l) and 6.9 +/- 6.2 micrograms/l, respectively. The Cp of OC was 1.08 +/- 0.3 ml/min and the MT of OC over 4-h dialysis exchange periods was 14.5 +/- 12.3 micrograms when using a dialysis solution containing 2.27% glucose. S-OC was significantly correlated with serum levels of alkaline phosphatase (r = 0.80), intact PTH (r = 0.82) and the MT of OC (r = 0.94). No significant correlations were found with serum levels of total calcium, phosphate, creatinine, total protein and dialytic age. These results suggest that the OC level in serum is influenced by both bone turnover and peritoneal clearance. Therefore, altered serum levels of OC should be interpreted always together with the peritoneal mass transfer of OC. Taking this into account, OC and intact PTH may be of value as markers of increased bone turnover secondary to renal osteodystrophy in CAPD.

Sugar transport in isolated rat kidney papillary collecting duct cells.

D-Glucose is an important substrate of energy metabolism and osmolyte synthesis in the renal papillary collecting duct. In order to characterize the cellular entry of D-glucose in this tubular segment, collecting duct cells were isolated from rat kidney papilla and the rate of D-glucose uptake was measured indirectly by monitoring the D-glucose-dependent O2 uptake in the presence of the uncoupler CCCP. D-Glucose uptake was found to be sodium-independent and not sensitive to phlorizin even at a concentration of 10(-3) M. Uptake was, however, completely inhibited by 10(-5) M cytochalasin B and 10(-4) M phloretin. The apparent Ki for cytochalasin B was 1.5 x 10(-6) M and for phloretin 2.0 x 10(-5) M. Studies on the substrate specificity revealed that at 1 mM D-mannose is taken up and metabolized to the same extent as D-glucose. A 50-fold higher concentration of 2-deoxy-D-glucose and 2-amino-2-deoxy-D-glucose inhibited D-glucose uptake completely whereas alpha-methyl-D-glucoside, D-allose, and D-galactose were without effect. Under conditions where D-glucose utilization was maximally stimulated an apparent Km of 1.2 mM and a Vmax of 1 mmol D-glucose/g protein.hour was found for D-glucose uptake. These results indicate that the D-glucose uptake into papillary collecting duct cells is probably mediated by a transport system similar to the one found in basal-lateral membranes of polarized renal, intestinal, and liver cells as well as in nonpolarized fat cells and erythrocytes.

Sorbitol metabolism in inner medullary collecting duct cells of diabetic rats.

Intracellular accumulation of sorbitol, generated from D-glucose via the aldose reductase pathway, is thought to play an important role in diabetic complications such as lens cataracts and neuropathy. In order to elucidate the effect of diabetes on the renal inner medulla, another sorbitol-rich tissue, male Wistar rats were treated with a single dose of streptozotocin (60 mg/kg body weight, i.p.). Six weeks later total inner medullary tissue (IM) or isolated inner medullary collecting duct (IMCD) cells were prepared. In diabetic IM tissue, sorbitol content was 1.8-fold higher than in control IM tissue (134 +/- 17 vs. 74 +/- 22 mumol/g tissue protein). Sorbitol production in both normal and diabetic IMCD cells was strongly dependent on extracellular D-glucose concentration. In normal cells, for example, sorbitol production was 90 +/- 9 mumol sorbitol/g protein x h at 45 mM D-glucose compared to 13 +/- 1 mumol/g protein x h at 5 mM. At identical D-glucose concentrations sorbitol synthesis in diabetic IMCD cells was, however, always significantly higher than in control cells (122% of control at 15 mM and 126% of control at 45 mM). In addition, aldose reductase activity in diabetic IM was found to be augmented. The maximal velocity was 4.2 times higher (97 +/- 22 U/g protein vs. 23 +/- 7 U/g protein) while the Km of the enzyme remained unchanged. Membrane permeability for sorbitol or the response to changes in extracellular osmolarity was not significantly different in diabetic IMCD cells and normal cells with correspondingly high intracellular sorbitol concentrations. Similarly the kinetic parameters of D-glucose uptake were not altered by streptozotocin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Osmotic regulation of the betaine metabolism in immortalized renal cells.

Betaine plays an important role in the osmoregulation of various renal cells. In the kidney betaine synthesis seems to be highest in the cortex, whereas osmotically regulated accumulation seems to play a crucial role in the inner medulla. Therefore, the influence of betaine synthesis on the long-term osmotic regulation of betaine content was investigated in epithelial SV40 transfected cell culture, derived from the outer medullary thick ascending limb of the loop of Henle (TALH) of rabbit kidney. Under hyperosmotic conditions the betaine content of TALH was significantly increased from 218 +/- 35 mumol/g protein (300 mOsm/liter; control) to 334 +/- 27 mumol/g (600 mOsm/liter; P < 0.0005). In addition the intracellular accumulation of 14C-betaine from 14C-choline was significantly elevated from 4.3 +/- 1.0 mumol/g protein x hr) to 8.2 +/- 1.0 mumol/g protein x hr; P < 0.001) under hyperosmotic conditions. Synthesis of betaine was also influenced by the extracellular betaine content. In a betaine free medium the synthesis of betaine was increased by 7% (300 mOsm/liter; NS) or 40% (600 mOsm/liter; P < 0.0001) when compared to betaine containing medium. The alteration of betaine synthesis is presumably caused by osmotic regulation of the betaine aldehyde dehydrogenase. Activity of this enzyme was significantly higher under hyperosmotic conditions compared to isoosmotic control conditions (Vmax 4.1 +/- 0.8 U/g protein; 600 mOsm/liter) versus 1.4 +/- 0.1 U/g (300 mOsm/liter; P < 0.0001), while the affinity to betaine aldehyde remained unaltered. These results demonstrate that during long-term adaptation, betaine synthesis in TALH cells of the outer medulla of rabbit kidney can be regulated by extracellular osmolarity.

Osmotic regulation of sorbitol in the thick ascending limb of Henle's loop.

Organic osmolytes, such as sorbitol, inositol, glycerophosphorylcholine, and betaine, play an important role in the osmoregulation of inner medullary cells of the kidney. The cells of the outer medulla are also exposed to elevated NaCl and urea concentrations during antidiuresis. Therefore, we investigated the mechanisms involved in the regulation of outer medullary organic osmolytes, especially cell sorbitol content of immortalized epithelial cells of the thick ascending limb of Henle's loop (TALH). In the cultured cell model, a 600 mosmol/l medium (osmolarity adjusted by addition of 150 mM NaCl or 300 mM sucrose) increased the intracellular sorbitol content significantly compared with a 300 mosmol/l medium. The accumulation of sorbitol appeared to be due to an increase of aldose reductase activity, which catalyzed sorbitol synthesis. Sorbitol degradation by sorbitol dehydrogenase was not detectable under our experimental conditions. After a sudden decrease of the extracellular osmolarity, the sorbitol permeability of the cellular membrane increased sevenfold within 10 min compared with isosmolar conditions. These results indicate that sorbitol, like inositol, plays an important role in the osmoregulation of TALH cells. Although the short-term regulation involves rapid changes in sorbitol membrane permeability, the longterm adaptation to low osmolarities is regulated by intracellular sorbitol synthesis.

Intracellular sorbitol content in isolated rat inner medullary collecting duct cells. Regulation by extracellular osmolarity.

In order to study the mechanisms involved in the regulation of renal inner medullary sorbitol content, collecting duct cells were isolated from rat inner medulla and the effect of extracellular osmolarity on sorbitol synthesis and sorbitol content was investigated. Cells isolated at 300 mosmol/l and incubated up to 24 h as primary cultures in 300 mosmol/l media or in media made 600 mosmol/l by the addition of 150 mM NaCl showed no difference in total synthesis. Intracellular sorbitol content was, however, 2.3-fold higher in the cells kept in the higher osmotic medium. Cells isolated at 600 mosmol/l released sorbitol about 8 times faster when transferred into hypoosmotic medium (300 mosmol/l) than when transferred into isoosmotic (600 mosmol/l) media. Cells exposed to hyperosmotic media (900 mosmol/l with NaCl) maintained a higher intracellular sorbitol content than cells incubated in isoosmotic media. Changes of intracellular sorbitol content could not be attributed entirely to cell lysis--as demonstrated by determination of cellular content of lactate and lactate dehydrogenase. The alteration in sorbitol membrane permeability was reversible and was only observed when poorly permeable solutes (such as NaCl and sucrose) were used for the experiments, changes in urea elicited no effect. It is proposed that rapid changes in membrane permeability to sorbitol play an important role in the adjustment of intracellular sorbitol concentration in inner medullary collecting duct cells to changes in extracellular osmolarity.

Regulation of ion content and cell volume in isolated rat renal IMCD cells under hypertonic conditions.

The adaptive responses of cell volume and intracellular inorganic electrolytes to hypertonicity were investigated in isolated inner medullary collecting duct (IMCD) cells in vitro. Intracellular water and element content were determined simultaneously by a rapid centrifugation technique and electron-probe microanalysis, respectively. In the absence of adenosine 3',5'-cyclic monophosphate (cAMP), cells isolated at 600 mosM (268 mM NaCl) shrank in 900 mosM buffer (418 mM NaCl) within 100 s to 79 +/- 2% of the control volume and showed no regulatory volume increase (RVI). K+ content decreased steadily during the first 5 min to 18% below control. K+ concentration initially increased 25 +/- 2% above control and was normalized after 5 min to 144 +/- 10 mM (n = 6). Na+ and Cl- content increased slowly during the 60-min incubation period; at this point intracellular Na+ and Cl- concentrations were 97 +/- 6 and 59 +/- 7% (n = 6) above control levels (75 +/- 12 mM Na+ and 143 +/- 29 mM Cl-), respectively. In the presence of 10(-4) M 8-bromo-cAMP (8-BrcAMP; used as a substitute for antidiuretic hormone), IMCD cells showed RVI within 5 min. K+ content initially dropped 18 +/- 2% below control and recovered during the next 4 min but was 12 +/- 4% below control after 60 min. K+ concentration showed similar changes. Na+ and Cl- content increased to a larger extent than in the absence of 8-BrcAMP to 95 +/- 10% (Na+) and 47 +/- 6% (Cl-) above control after 60 min, whereas Na+ and Cl- concentrations were the same as without 8-BrcAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Ion content and cell volume in isolated collecting duct cells: effect of hypotonicity.

On isolated inner medullary collecting duct (IMCD) cells of the rat kidney the capability of osmoregulatory adaptation was investigated in vitro. IMCD cells were isolated by differential centrifugation at 600 mOsm (268 mM NaCl) and subsequently exposed to hypotonic buffers (300 mOsm, 118 mM NaCl). The alterations of ion content and cell volume following this change in extracellular osmolarity were studied by electron probe microanalysis and determination of intracellular water. After swelling within 40 seconds to 152 +/- 15% of control (P < 0.001; N = 9) cell volume was restored after 15 minutes. This regulatory volume decrease (RVD) was observed irrespective whether extracellular osmolarity was changed by using NaCl or mannitol as the major osmolyte. During RVD the cells lost sodium (48 +/- 11%) and chloride (14 +/- 5%), and the potassium content remained nearly unchanged. Correspondingly, sodium and chloride concentrations were progressively lowered, whereas the potassium concentration changed only transiently. RVD was diminished by 10(-4) M NPPB, 10(-3) M SITS and in the absence of HCO3-. Twenty millimoles of ouabain or 5 mM barium also inhibited RVD with little additive effect. A total of 10(-3) M amiloride and 10(-4) M bumetanide showed no effect on the hypoosmotic volume response. The experiments show that in isolated IMCD cells exposed to hypotonic conditions, rapid reversible changes in cell volume and sustained alterations in cell inorganic ion content occur, and thereby transmembrane sodium and potassium gradients are maintained. Since the loss in inorganic electrolytes does not account for RVD, the major part of volume regulation seems to occur via changes in organic osmolytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathways for the synthesis of sorbitol from 13C-labeled hexoses, pentose, and glycerol in renal papillary tissue.

Suspensions of rabbit renal papillary tissue were incubated with D-[6-13C]glucose, D-[1-13C]fructose, D-[1-13C]ribose, and [2-13C]glycerol. The perchloric acid extracts of the above incubations were investigated with 13C NMR spectroscopy. All 13C-labeled substrates give rise to 13C-labeled D-sorbitol. D-[6-13C]Glucose and D-[1-13C]fructose are converted directly into D-sorbitol via the aldose reductase and sorbitol dehydrogenase pathway, respectively, whereas D-[1-13C]ribose and [2-13C]glycerol give rise to labeling of the D-glyceraldehyde pool which on its turn causes a labeling of D-sorbitol. Label exchanges observed from incubations with glycerol and D-ribose indicate that the pentose shunt plays a role in this synthesis of D-sorbitol.

Renal inner medullary sorbitol metabolism.

Sorbitol participates in the osmoregulation of several renal cells and has also been found in isolated inner medullary collecting duct (IMCD) cells in primary culture. Therefore, osmotic regulation and distribution of sorbitol and the key enzymes of sorbitol metabolism, aldose reductase and sorbitol dehydrogenase in the renal inner medulla, were investigated in vivo under various osmotic conditions (control, diuresis, antidiuresis). In homogenates of the renal inner medulla of Wistar rats, the sorbitol content correlated with the urine osmolarity [68 +/- 12 mumol/g protein (control), 28 +/- 9 mumol/g (diuresis), 110 +/- 15 mumol/g (antidiuresis)]. Similar results were obtained for the activity of aldose reductase (sorbitol synthesis) [25 +/- 4 U/g (control), 19 +/- 3 U/g (diuresis), and 48 +/- 7 U/g (antidiuresis)]. On the contrary, the activity of sorbitol dehydrogenase (sorbitol degradation) was significantly increased to 1.26 +/- 0.42 U/g under diuretic conditions vs. control (0.84 +/- 0.14 U/g, P < 0.05). These results demonstrate the correlation between the enzymes of sorbitol synthesis and sorbitol degradation in the intact inner medulla and the urine osmolarity in vivo. Whereas the aldose reductase activity was 2.3-fold enriched in IMCD cells, the specific activity of sorbitol dehydrogenase was relatively increased in a preparation of enriched interstitial cells. This distribution was not dependent on the various diuretic conditions. These results indicate that enzymes of synthesis and of degradation of sorbitol are osmotically regulated in vivo. Therefore, the enzymatic activities of sorbitol synthesis appear to be primarily located in epithelial cells, whereas enzymatic activities of sorbitol degradation seem to be localized in interstitial cells of the renal inner medulla.

Choline transport and its osmotic regulation in renal cells derived from the rabbit outer medullary thick ascending limb of Henle.

Organic osmolytes such as betaine and glycerophosphorylcholine (GPC) are of major importance concerning volume regulation of inner and outer medullary epithelial cells. Recently we demonstrated that the intracellular betaine content in rabbit kidney cells derived from the outer medullary thick ascending limb of Henle's loop (TALH) is osmotically regulated by betaine synthesis. In this context it was our purpose to characterize the uptake of choline, a precursor of betaine and GPC. We found TALH cells to possess a specific choline transport system with a maximum velocity (Vmax) of 71 +/- 12 pmol . micro l-1 cell water . min-1 and an apparent affinity (Km) of 155 +/- 19 micromol . l-1. The uptake of choline was sodium independent and not electrogenic, but it was significantly reduced by the removement of chloride from the incubation medium. After long-term adaptation of TALH cells to a hyperosmotic medium (600 mosmol . l-1, osmolarity adjusted with NaCl or urea) a significant higher choline uptake rate was observed (Vmax: 166 +/- 9 (NaCl), 96 +/- 12 (urea) pmol . microl-1 cell water . min-1). Our results suggest that the uptake of choline is due to higher intracellular requirements of choline under hypertonic conditions. Finally, an increase in the Vmax of the choline transport system may enable sufficient synthesis of betaine and GPC.

Characteristics of urea transport of cells derived from rabbit thick ascending limb of Henle's loop.

BACKGROUND: The thick ascending limb of Henle's loop (TALH) is thought to be involved in the regulation of the renal urea gradient. METHODS: We have characterized the uptake of urea (oil density centrifugation and 2-compartment-culture) and volume regulation (impedance measurement) in highly differentiated cells derived from rabbit outer medulla. RESULTS: TALH cells exposed to 600 mOsm/liter (300 mM urea) shrunk to 72 +/- 5% of the isoosmotic volume. Due to a regulatory volume increase (RVI), the cell volume was almost completely regained at 92 +/- 6% after five minutes. The uptake of 14C-urea in the presence of urea concentrations up to 600 mM did not show any saturation. In the presence of phloretin the urea uptake decreased to 69 +/- 14%. The transport was sodium and chloride independent. Changing the membrane potential caused an increase of regulatory volume increase and urea uptake. Hyperosmolarity induced by sucrose (300 mM) and NaCl (150 mM) caused a decrease of urea uptake to 70 +/- 14% and 53 +/- 11%, respectively. The permeability coefficient (P) in a two compartment culture was P = 1.7 . 10(-6) +/- 0.39.10(-6) cm/second, suggesting a relatively low permeability. CONCLUSION: Due to the low permeability, it seems impossible to achieve a physiologically significant participation of the TALH in the urea circulation within the nephron. However, the results of this study provides significant hints about the existence of a specific urea transport mechanism that enables the cell to adapt rapidly to different osmolarities.

[Study of kidney function using isolated cells]. / Untersuchung der Nierenfunktion mit Hilfe isolierter Zellen.

After summarizing the progress which has been made with regard to the isolation and characterization of homogeneous cell populations from the kidney, a brief survey of current techniques available for the analysis of intracellular parameters is given. Special emphasis is thereby placed on the use of electron probe X-ray microanalysis to determine intracellular elements and on "in vivo" nuclear magnetic resonance to define metabolic pathways in isolated cells. These methods have been applied to study ion and substrate fluxes in isolated collecting duct cells and the response of these cells to changes in osmolality of the extracellular medium. This response involves initially fast water movements accompanied by changes in intracellular sodium and chloride but not potassium concentration. Longterm adaptation is achieved by the adjustment of the intracellular concentration of "organic osmolytes" such as sorbitol, myoinositol, glycerophosphorylcholine, and betaine through changes in the rate of efflux of these metabolites from the cell. In the last section the effect of experimentally induced diabetes mellitus on the osmoregulation in isolated collecting ducts is described.