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.

Human renal fibroblasts derived from normal and fibrotic kidneys show differences in increase of extracellular matrix synthesis and cell proliferation upon angiotensin II exposure.

Angiotensin II (Ang II) as a vasoactive hormone may be involved in progressive renal interstitial fibrosis. We investigated the influence of Ang II on cell proliferation and synthesis of extracellular matrix (collagen I, III and fibronectin) in human renal fibroblasts derived from normal (TK 173 cell line) and fibrotic (TK 188 cell line) kidneys which possess both Ang II type l and type 2 (AT1 and AT2) receptors. Incubation of the cells with Ang II increased the cell proliferation and the synthesis of extracellular matrix significantly in both cell lines. However, proliferation and extracellular matrix synthesis showed a greater increase in the cells derived from the fibrotic kidney. The Ang II mediated effect on cell proliferation and extracellular matrix synthesis was diminished in the presence of the AT1 receptor blocker losartan in both cell lines. No inhibition was observed using the AT2 receptor blocker PD123319. Ang II induced cell proliferation could be completely inhibited by incubation with human TGF-beta1 antibody. Incubation with Ang II did not affect TGF beta 1 production but in untreated cells TGF-beta 1 content was higher in the cells derived from the fibrotic kidney. This might be the reason for the more sensitive reaction on exposure to Ang II.

First steps toward the establishment of a German low-density lipoprotein-apheresis registry: recommendations for the indication and for quality management.

New recommendations for the indication of treatment with selective extracorporeal plasma therapy low-density lipoprotein apheresis (LDL-apheresis) in the prevention of coronary heart disease are urgently needed. The following points are the first results of the ongoing discussion process for indications for LDL-apheresis in Germany: all patients with homozygous familial hypercholesterolemia with functional or genetically determined lack or dysfunction of LDL receptors and plasma LDL cholesterol levels >13.0 mmol/L (>500 mg/dL); patients with coronary heart disease (CHD) documented by clinical symptoms and imaging procedures in which over a period of at least 3 months the plasma LDL cholesterol levels cannot be lowered below 3.3 mmol/L (130 mg/dL) by a generally accepted, maximal drug-induced and documented therapy in combination with a cholesterol-lowering diet; and patients with progression of their CHD documented by clinical symptoms and imaging procedures and repeated plasma Lp(a) levels >60 mg/dL, even if the plasma LDL cholesterol levels are lower than 3.3 mmol/L (130 mg/dL). Respective goals for LDL cholesterol concentrations for high-risk patients have been recently defined by various international societies. To safely put into practice the recommendations for LDL-apheresis previously mentioned, standardized treatment guidelines for LDL-apheresis need to be established in Germany that should be supervised by an appropriate registry.

Polarized function of thick ascending limbs of Henle cells in osmoregulation.

BACKGROUND: Organic osmolytes are necessary for osmoregulation in mammalian kidney. Since renal epithelial cells in many cases possess specific mechanisms both for uptake and osmotically regulated release, we investigated their localization in polarized cells. METHODS: An immortalized epithelial cell line derived from the thick ascending limb of Henle's loop (TALH) was used to examine the transport characteristics of the apical and basolateral plasma membranes for osmotic regulation of organic osmolytes. Cells were cultured on filters in a two-compartment chamber. RESULTS: In culture under hypertonic conditions the TALH cells accumulated in the following balance: sorbitoverline> betaine = myo-inositoverline> glycerophosphoryl choline (GPC). When extracellular osmolarity was decreased, then sorbitol was released on the apical side, whereas betaine and myo-inositol efflux occurred on the basolateral side. GPC release showed no preference of either side. Taurine did not seem to be necessary for osmoregulation under these conditions. Osmotically regulated myo-inositol and betaine uptake was located on the apical side, and choline uptake took place on both sides equally. CONCLUSION: These results show that in renal epithelial cells, both osmotically induced release and the uptake of organic osmolytes are divided between the apical and the basolateral sides. This might be important for volume regulation.

Evidence for a sorbitol transport system in immortalized human renal interstitial cells.

Sorbitol plays a major role in the maintenance of cell volume and functional integrity of several renal cells. Sorbitol synthesis takes place in inner collecting duct cells, whereas sorbitol dehydrogenase activity, which catalyzes the degradation of sorbitol to fructose, could mainly be detected in renal inner medullary interstitial cells. Therefore, we supposed that interstitial cells would require a sorbitol transport into the cells. However, such a transport system has not yet been described. Therefore, we have characterized the uptake of sorbitol in immortalized interstitial TK-173 cells, which were derived from human renal fibroblasts. Comparable to fresh isolated renal fibroblasts of the rat, immortalized TK-173 cells have a high sorbitol dehydrogenase activity. In this report, a temperature-dependent sorbitol uptake with saturation kinetics could be detected in immortalized TK-173 cells. The transport is characterized by a high velocity (Vmax 84 mmol/l x h) and an apparent Km of 10 mmol/l. The sorbitol uptake is independent of membrane potential, sodium, and chloride. Altogether, the physiological characteristics of this sorbitol transport are different from those of the osmotically regulated sorbitol efflux from epithelial cells. These results provide evidence that TK-173 cells derived from renal fibroblasts have a specific sorbitol transport. Furthermore, these data suggest a cooperation between epithelial and interstitial cells concerning osmoregulation.

Characterization of aldose reductase from the thick ascending limb of Henle's loop of rabbit kidney.

BACKGROUND: The organic osmolyte sorbitol plays an important role in the osmoregulation of immortalized epithelial cells of the thick ascending limb of Henle's loop (TALH) of rabbit. The intracellular sorbitol content seems to depend strongly on the extracellular osmolarity. To investigate the nature of the osmotic regulation we characterized the aldose reductase. METHODS: We determined aldose reductase activity enzymatically and the content of organic osmolytes by HPLC. RESULTS: The aldose reductase activity correlates with the extracellular tonicity. Elevating the osmolarity of the medium from 300 to 600 mosm/l by addition of NaCl or sucrose resulted in a significant increase of maximal velocity (V(max)) of the adapted cells from 8 +/- 1 micromol/g x min (300 mosm/l) to 322 +/- 28 micromol/g x min (600 mosm/l, NaCl) or 54 +/- 9 micromol/g x min (600 mosm/l, sucrose), respectively, while affinity (K(m)) remained unchanged. But we found no rise of aldose reductase activity when extracellular urea concentration was elevated. Similar alterations in V(max) were observed when the activity of the highly enriched enzyme was determined with glucose as substrate. Elevation of the extracellular osmolarity by NaCl and sucrose strongly induced the expression of aldose reductase protein with an apparent molecular weight of 39 kD. The affinity of glucose is characteristically low with a K(m) above 300 mmol/l. Aldose reductase utilizes both NADPH and with lower affinity NADH as coenzymes. In vitro sulfate ions (0.4 mol/l) results in a two-fold activation of the aldose reductase activity whereas sodium (200-400 mmol/l) decreased the activity significantly (22-33%). Potassium and chloride up to 400 mmol/l did not alter the aldose reductase activity in vitro. CONCLUSIONS: These results indicate that the aldose reductase of TALH cells of the outer medulla is osmotically regulated and has many similarities with aldose reductase in renal inner medulla. Therefore, intracellular sorbitol synthesis seems to be of similar importance in the osmoregulation of TALH cells as in the inner medulla.

Cisplatin nephrotoxicity in children after continuous 72-h and 3x1-h infusions.

Little is known about the association between the rate of cisplatin administration and the severity of cisplatin-induced renal damage in children. The purpose of this study was to compare severity and reversibility of renal damage in children after continuous and repetitive bolus administration of cisplatin and to correlate these data with pharmacokinetic parameters. Study subjects included six children (ten courses) receiving cisplatin as 1-h bolus infusions on three consecutive days (3x40 mg/m2) and four children (eight courses) receiving 72-h continuous infusions (120 mg/m2). In all courses, signs of glomerular and tubular damage were seen, as evidenced by elevated urinary excretion of alpha1-microglobulin, albumin and N-acetyl-beta-D-glucosaminidase and decreased glomerular filtration rate (GFR). Comparing the two infusion regimens, the 1-h bolus administration of cisplatin was followed by significantly higher peak free platinum concentrations in plasma and urine (P<0.001), resulting in lower nadirs of the GFR (P<0.005). Correlations were found between both peak free platinum concentrations in plasma and urine and maxima of urinary albumin and N-acetyl-beta-D-glucosaminidase excretion. Within 12 months after completion of cisplatin therapy, children in the 1-h bolus group had recovered only partially from subclinical nephrotoxicity, with five out of six showing pathological proteinuria. The results provide clear evidence that long-term ciplatin infusions are less nephrotoxic than repetitive bolus infusions.

Reduction of hepatitis C virus load by H.E.L.P.-LDL apheresis.

The association of HCV with apolipoprotein B containing lipoproteins has been observed and this led to the assumption that the LDL receptor may also serve as a candidate receptor for HCV. H.E.L.P.-LDL apheresis is suggested to be an effective and rapid tool to safely eliminate apolipoprotein B containing lipoproteins. In this pilot study, we have investigated whether H.E.L.P. treatment would reduce HCV load in five patients, all infected for more than 4 years with HCV and resistant against established anti-HCV therapy (interferon, ribaverin). HCV-RNA was determined by RT-PCR in plasma immediately before the start of apheresis (SA) and after treatment of 2500 mL plasma (AA). H.E.L.P. apheresis led to a mean decrease of 77.3% (16th percentile 36.5%, 84th percentile 89.6%) of HCV-RNA when AA values were compared to SA values. This decline was reproducible during nine treatment procedures, but was not correlated to the decrease in LDL cholesterol. This investigation shows for the first time that HCV load can be reduced by H.E.L.P. apheresis, which is an established and approved therapy for hypercholesterolemia. Even though the efficiency of viral load reduction varied between single procedures and did not correlate to LDL removal, this extracorporeal therapy opens the possibility to treat patients with established immune modulatory and antiviral therapy in the interval between two apheresis procedures.

Volume regulation of thick ascending limb of Henle cells: significance of organic osmolytes.

The thick ascending loop of Henle (TALH) is exposed to high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. Therefore, the volume regulation of TALH cells, derived from the TALH loop of rabbit kidneys, was analyzed. The volume was determined by impedance measurements. TALH cells, which were adapted to different osmolarities (300 and 600 mosm/l), showed no significant differences in their cell volume. Therefore, a complete volume regulation could be supposed. An increase in extracellular osmolarity from 300 to 600 mosm/l (osmolarity adjusted by addition of 150 mM NaCl) immediately led to a reduction in the cell volume by 37 +/- 6% (n = 6). A regulatory volume increase (RVI) was not observed within 10 min but after 24 h. Conversely, a sudden cell swelling by 44 +/- 5% (n = 4) was detected within 20 s following an extracellular hypoosmotic challenge (from 600 to 300 mosm/l). The subsequent volume regulatory decrease (RVD) required a period of 7 days. Specific inhibitors of important ion transporters had no effect on volume regulation. Thus, changes in the ion conductivity do not seem to influence the processes of RVI and RVD. Conversely, the intracellular content of the organic osmolytes, sorbitol, inositol, betaine, and glycerophosphorylcholine, changed in the course of RVI and RVD. These results provide evidence that TALH cells are capable of maintaining their volume despite large extracellular osmotic changes. RVI and RVD are mainly regulated by changes in the intracellular content of organic osmolytes within 1 and 7 days.

Immunocytological determination of lymphocytes and monocytes/macrophages in urinary sediments of renal allograft recipients.

BACKGROUND: Urinary studies using Papanicolaou staining following kidney transplantation led to the conjecture that acute allograft rejection might be accompanied by an increased lymphocyturia. However, it is difficult to distinguish lymphoid cells from other urinary cells using conventional stains. METHODS: Staining of urinary lymphocytes using FITC-labelled antibodies is complicated by a high unspecific fluorescence that limits the evaluation. Therefore, we developed a method to stain urinary lymphocytes using enzyme-linked antibodies. The cells were cytocentrifuged onto microscope slides and were fixed. RESULTS: By means of a combined evaluation of Papanicolaou and immunocytochemical staining, CD3-positive pan T cells, CD4-positive T-helper cells, CD8-positive cytotoxic/suppressor cells, and CD14-positive monocytes/macrophages of urinary sediments were determined in 41 kidney graft recipients following renal transplantation. During periods of normal graft function, neither positive lymphocytes nor positive monocytes/macrophages were found in the urinary sediments. However, in the course of acute allograft rejection a significant increase in positive lymphocytes and positive monocytes/macrophages could be observed. Interestingly, in cases of acute allograft rejection the distribution of urinary lymphocytes and monocytes was comparable to the distribution of infiltrating immunocompetent cells in renal allograft biopsies. CONCLUSION: The present study demonstrates that immunocytochemical staining via enzyme-conjugated antibodies is a reliable method to visualize T lymphocytes and monocytes/macrophages in the urinary sediment, and that this technique may be of special diagnostic value in the diagnosis of acute allograft rejection.

Soluble CD-4 and CD-8 as markers of immunological activation in renal transplant recipients.

BACKGROUND: T lymphocytes are activated following kidney transplantation in cases of acute graft rejection and viral infections. In plasma, elevated levels of T-cell markers can be measured in soluble form. The reason for this shedding is still not entirely understood. METHODS: Plasma concentrations of soluble CD-4 and CD-8 (sCD-4, sCD-8) were determined in 78 patients following kidney transplantation by commercially available enzyme-linked immunosorbent assay (ELISA) test kits. RESULTS: The concentrations of both soluble T-cell markers increased significantly in the course of acute allograft rejections and cytomegalovirus (CMV) infections. Frequently, the parameters increased shortly before clinical diagnosis and decreased under successful therapy. Additionally, sCD-8 showed significant higher plasma concentrations in cases of CMV infection as compared with acute allograft rejections. Accordingly, the sCD-4/sCD-8 ratio increased in cases of acute allograft rejection and decreased during CMV infections. Cyclosporin A nephrotoxicity caused no significant changes in the sCD-4 and sCD-8 levels in plasma. CONCLUSION: The present study demonstrates that sCD-4 and sCD-8 are markers of immunological activation and may enable a further differentiation of T-cell activation if serial measurements are performed. However, further prospective investigations are necessary to elucidate the diagnostic potential of sCD-4 and sCD-8 for monitoring acute rejection and viral infection in kidney graft recipients.

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.

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.

Differentiating and proliferative effects of HGF in renal proximal tubular cells are mediated via different signalling pathways.

BACKGROUND: As a renotropic cytokine, hepatocyte growth factor (HGF) prevents acute renal failure and accelerates renal regeneration. HGF initiates its biological effects by interaction with specific transmembrane receptors, the c-Met proto-oncogene, possessing an intracellular tyrosine kinase domain. We tested the hypothesis of whether the complex biological effects of HGF in renal proximal tubular cells are mediated by different intracellular signalling cascades and/or different receptors. METHODS: PT-1 cells, a proximal tubular cell line derived from rabbit kidney, were cultured under defined serum-free conditions to examine the biological effects of exogenously added HGF. By specific assays, we determined HGF binding and its effects on cell proliferation, migration, scattering and tubulogenic differentiation. To investigate whether HGF action could be inhibited by protein tyrosine kinase inhibitors (PTKIs), cells were incubated with HGF and different concentrations of herbimycin A, genestein, methyl-2,5-dihydroxycinnamate (MDC) and geldanamycin. All PTKIs are known inhibitors of pp60(c-src), a non-receptor tyrosine kinase involved in cell growth control. RESULTS: HGF bound with high affinity to cell membrane receptors and displayed multiple biological effects. Compared with serum-free controls, HGF increased the number of microvilli 1.5-fold, enhanced cell proliferation and migration 1.8-fold, and stimulated the formation of tubular structures 2.3-fold. Consistent with the known tyrosine kinase activity of the c-Met receptor, the mitogenic and motogenic effects of HGF were inhibited by PTKIs in a dose-dependent manner with the following order of potency: geldanamycin > herbimycin A > genestein > MDC. In contrast, however, the HGF-induced tubulogenic cell differentiation was not inhibited specifically by PTKIs. CONCLUSIONS: The finding that PTKIs inhibited the mitogenic response but not the tubulogenic differentiation induced by HGF indicates different intracellular signal transduction pathways. We suggest that pp60(c-src) plays a key role in mediating the mitogenic and motogenic action of HGF, whereas tubulogenic cell differentiation induced by HGF is transduced by a pp60(c-src)-independent signalling pathway.

Rat renal expression of mRNA coding for aldose reductase and sorbitol dehydrogenase and its osmotic regulation in inner medullary collecting duct cells.

Sorbitol plays an important role in the osmoregulation of several renal cell types, especially the inner medullary collecting duct (IMCD) cells. Very little information is available concerning the expression of the enzymes of sorbitol metabolism (aldose reductase (AR) and sorbitol dehydrogenase (SDH)) on the RNA level under different osmotic conditions. We employed a RT-PCR-based strategy to investigate the regulation of mRNA coding for AR and SDH. For AR two primers (derived from the sequence of the rat eye lens) were chosen which amplify a 668-bp product. For SDH (considering the sequence of rat liver) three primers were chosen, amplifying a 367- and a 1, 068-bp fragment. Digestion with restriction enzymes and sequencing of the products clearly indicate that the specific mRNA of AR and SDH was amplified. By relative quantitative determination of the amplification products a more than 4-fold increase in mRNA for AR in IMCD cells was observed within 24 h after increasing the extracellular osmolarity from 600 to 900 mosm/l. Decreasing the osmolarity from 600 to 300 mosm/l resulted in a reduction in the mRNA level by 70%. The complete adaptation of the AR activity needed 3 (increasing osmolarity) and 6 days (decreasing osmolarity). Osmotically induced alterations in the levels of mRNA coding for SDH could not be observed. These results suggest that the adaptation of sorbitol synthesis occurs by a rapid regulation of transcription or stability of specific mRNA. For a complete synthesis or degradation of AR 3-6 days are necessary. Thus sorbitol synthesis in IMCD is more rapidly adapted to increasing osmolarities than to decreasing osmolarities.

Biochemistry and physiology of carbohydrates in the renal collecting duct.

Using 13C-NMR analysis of cell extracts, enzymatic determination of metabolites and cofactors as well as enzyme assays on cell homogenates aerobic and anaerobic glycolysis, sorbitol formation by aldose reductase, the pentose phosphate shunt, and gluconeogenesis could be identified as the major pathways of D-glucose metabolism in renal inner medullary collecting ducts. In flux studies it was shown that D-glucose enters the collecting duct cells via a sodium-independent, cytochalasin- and phloretin-inhibitable transport system located at the basal-lateral cell side. At the same side sorbitol leaves the cells during regulatory volume decrease in a calcium-calmodulin-dependent fashion. From cell isolation studies it is proposed that sorbitol is taken up by adjacent (interstitial) cells, converted into fructose and then recycled to the collecting duct cells. This cycle might prevent carbohydrate wasting. Thus, IMCD cells exhibit unique aspects of carbohydrate biochemistry and physiology which enable them to function in a surrounding of low oxygen tension, low substrate supply, and extreme changes in extracellular osmolality.