Comparison and commutability study among four faecal immunochemical tests (FIT) systems.

OBJECTIVES: Faecal immunochemical tests for haemoglobin (FIT) are used in colorectal cancer screening programs around the world and increasingly for triage of symptomatic patients. FIT results are currently not traceable to a common reference standard and results obtained on various FIT systems may not be equivalent. The size of the bias between the systems is difficult to quantify due to the complex pre-analytical aspects of FIT. METHODS: This study aimed to quantify the bias and the correlation between four FIT systems by measuring a panel of 38 faecal samples while limiting the effect of the pre-analytical aspects. In addition, the commutability of seven candidate reference materials (RM) was assessed. RESULTS: Pairwise method comparisons based on faecal samples demonstrated Pearson correlation coefficients ranging between 0.944 and 0.970 and an average proportional bias of -30 to -35 % for one FIT system compared to the other three. The relative standard deviation among biases of the individual samples was around 20 %. Due to these sample specific differences, no decisive conclusions could be drawn in the commutability study. However, two candidate RMs, prepared in the FIT system-specific storage/extraction buffers, had a better commutable profile than the other five. CONCLUSIONS: The use of a common threshold for all FIT systems is currently not possible due to the presence of a proportional bias. We have identified potential commutable RMs to take to further studies on the production of a common calibrator, with the aim being to reduce the analytical bias observed on different FIT systems.

Recommendations for Setting a Criterion for Assessing Commutability of Secondary Calibrator Certified Reference Materials.

A secondary higher-order calibrator is required to be commutable with clinical samples to be suitable for use in the calibration hierarchy of an end-user clinical laboratory in vitro diagnostic medical device (IVD-MD). Commutability is a property of a reference material that means results for a reference material and for clinical samples have the same numeric relationship, within specified limits, across the measurement procedures for which the reference material is intended to be used. Procedures for assessing commutability have been described in the literature. This report provides recommendations for establishing a quantitative criterion to assess the commutability of a certified reference material (CRM). The criterion is the maximum allowable noncommutability bias (MANCB) that allows a CRM to be used as a calibrator in a calibration hierarchy for an IVD-MD without exceeding the maximum allowable combined standard uncertainty for a clinical sample result (umaxCS). Consequently, the MANCB is derived as a fraction of the umaxCS for the measurand. The suitability of an MANCB for practical use in a commutability assessment is determined by estimating the number of measurements of clinical samples and CRMs required based on the precision performance and nonselectivity for the measurand of the measurement procedures in the assessment. Guidance is also provided for evaluating indeterminate commutability conclusions and how to report results of a commutability assessment.

Recommendations for Setting a Criterion and Assessing Commutability of Sample Materials Used in External Quality Assessment/Proficiency Testing Schemes.

It is important for external quality assessment materials (EQAMs) to be commutable with clinical samples; i.e., they should behave like clinical samples when measured using end-user clinical laboratory in vitro diagnostic medical devices (IVD-MDs). Using commutable EQAMs makes it possible to evaluate metrological traceability and/or equivalence of results between IVD-MDs. The criterion for assessing commutability of an EQAM between 2 IVD-MDs is that its result should be within the prediction interval limits based on the statistical distribution of the clinical sample results from the 2 IVD-MDs being compared. The width of the prediction interval is, among other things, dependent on the analytical performance characteristics of the IVD-MDs. A presupposition for using this criterion is that the differences in nonselectivity between the 2 IVD-MDs being compared are acceptable. An acceptable difference in nonselectivity should be small relative to the analytical performance specifications used in the external quality assessment scheme. The acceptable difference in nonselectivity is used to modify the prediction interval criterion for commutability assessment. The present report provides recommendations on how to establish a criterion for acceptable commutability for EQAMS, establish the difference in nonselectivity that can be accepted between IVD-MDs, and perform a commutability assessment. The report also contains examples for performing a commutability assessment of EQAMs.

Albumin determined by bromocresol green leads to erroneous results in routine evaluation of patients with chronic kidney disease.

OBJECTIVES: Measurement of plasma albumin is pivotal for clinical decision-making in patients with chronic kidney disease (CKD). Routinely used methods as bromocresol green (BCG) and bromocresol purple (BCP) can suffer from aselectivity, but the impact of aselectivity on the accuracy of plasma albumin results of CKD-patients is still unknown. Therefore, we evaluated the performance of BCG-, BCP- and JCTLM-endorsed immunological methods in patients with various stages of CKD. METHODS: We evaluated the performance of commonly used albumin methods in patients with CKD stages G1 through G5, the latter divided in two groups based on whether they received hemodialysis treatment. In total, 163 patient plasma samples were measured at 14 laboratories, on six different BCG and BCP-platforms, and four different immunological platforms. The results were compared with an ERM-DA-470k-corrected nephelometric assay. The implications on outcome is evaluated by the proportion of patient results <38 g/L for the diagnosis of protein energy wasting. RESULTS: Albumin results determined with BCP- and immunological methods showed the best agreement with the target value (92.7 and 86.2 %, respectively vs. 66.7 % for BCG, namely due to overestimation). The relative agreement of each method with the target value was platform-dependent, with larger variability in agreement between platforms noted for BCG and immunological methods (3.2-4.6 and 2.6-5.3 %) as opposed to BCP (0.7-1.5 %). The stage of CKD had similar effects on the variability in agreement for the three method-groups (0.6-1.8 % vs. 0.7-1.5 % vs. 0.4-1.6 %). The differences between methods cause discrepancies in clinical decision-making, as structurally fewer patients were diagnosed with protein energy wasting upon using BCG-based albumin results. CONCLUSIONS: Our study shows that BCP is fit for the intended use to measure plasma albumin levels in CKD patients from all stages, including patients on hemodialysis. In contrast, most BCG-based platforms falsely overestimate the plasma albumin concentration.

Diagnostic methods for neonatal hyperbilirubinemia: benefits, limitations, requirements, and novel developments.

Invasive bilirubin measurements remain the gold standard for the diagnosis and treatment of infants with severe neonatal hyperbilirubinemia. The present paper describes different methods currently available to assess hyperbilirubinemia in newborn infants. Novel point-of-care bilirubin measurement methods, such as the BiliSpec and the Bilistick, would benefit many newborn infants, especially in low-income and middle-income countries where the access to costly multi-analyzer in vitro diagnostic instruments is limited. Total serum bilirubin test results should be accurate within permissible limits of measurement uncertainty to be fit for clinical purposes. This implies correct implementation of internationally endorsed reference measurement systems as well as participation in external quality assessment programs. Novel analytic methods may, apart from bilirubin, include the determination of bilirubin photoisomers and bilirubin oxidation products in blood and even in other biological matrices. IMPACT: Key message: Bilirubin measurements in blood remain the gold standard for diagnosis and treatment of severe neonatal hyperbilirubinemia (SNH). External quality assessment (EQA) plays an important role in revealing inaccuracies in diagnostic bilirubin measurements. What does this article add to the existing literature? We provide analytic performance data on total serum bilirubin (TSB) as measured during recent EQA surveys. We review novel diagnostic point-of-care (POC) bilirubin measurement methods and analytic methods for determining bilirubin levels in biological matrices other than blood. Impact: Manufacturers should make TSB test results traceable to the internationally endorsed total bilirubin reference measurement system and should ensure permissible limits of measurement uncertainty.

Screening methods for neonatal hyperbilirubinemia: benefits, limitations, requirements, and novel developments.

Severe neonatal hyperbilirubinemia (SNH) is a serious condition that occurs worldwide. Timely recognition with bilirubin determination is key in the management of SNH. Visual assessment of jaundice is unreliable. Fortunately, transcutaneous bilirubin measurement for screening newborn infants is routinely available in many hospitals and outpatient settings. Despite a few limitations, the use of transcutaneous devices facilitates early recognition and appropriate management of neonatal jaundice. Unfortunately, however, advanced and often costly screening modalities are not accessible to everyone, while there is an urgent need for inexpensive yet accurate instruments to assess total serum bilirubin (TSB). In the near future, novel icterometers, and in particular optical bilirubin estimates obtained with a smartphone camera and processed with a smartphone application (app), seem promising methods for screening for SNH. If proven reliable, these methods may empower outpatient health workers as well as parents at home to detect jaundice using a simple portable device. Successful implementation of ubiquitous bilirubin screening may contribute substantially to the reduction of the worldwide burden of SNH. The benefits of non-invasive bilirubin screening notwithstanding, any bilirubin determination obtained through non-invasive screening must be confirmed by a diagnostic method before treatment. IMPACT: Key message: Screening methods for neonatal hyperbilirubinemia facilitate early recognition and timely treatment of severe neonatal hyperbilirubinemia (SNH). Any bilirubin screening result obtained must be confirmed by a diagnostic method. What does this article add to the existing literature? Data on optical bilirubin estimation are summarized. Niche research strategies for prevention of SNH are presented. Impact: Transcutaneous screening for neonatal hyperbilirubinemia contributes to the prevention of SNH. A smartphone application with optical bilirubin estimation seems a promising low-cost screening method, especially in low-resource settings or at home.

Evaluation of the ARKRAY HA-8190V instrument for HbA1c.

OBJECTIVES: Hemoglobin A1c (HbA1c) is a valuable parameter in the monitoring of diabetic patients and increasingly in diagnosis of diabetes. Manufacturers continuously optimize instruments, currently the main focus is to achieve faster turnaround times. It is important that performance specifications remain of high enough standard, which is evaluated in this study for the new ARKRAY HA-8190V instrument. METHODS: The Clinical and Laboratory Standards Institute (CLSI) protocols EP-5, EP-9 and EP-10 were applied to investigate imprecision, bias and linearity. In addition potential interferences, performance in External Quality Assessment (EQA) and performance against the HA-8180V instrument in 220 clinical samples was evaluated. RESULTS: The HA-8190V demonstrates a CV of ≤0.8% in IFCC SI units (≤0.6% National Glycohemoglobin Standardization Program [NGSP]) at 34 and 102 mmol/mol levels (5.3 and 11.5% NGSP) and a bias of -0.1 mmol/mol (-0.01% NGSP) at a concentration of 50 mmol/mol (6.7% NGSP), but with a significant slope as compared to target values. This results in a bias of -1.0 and 0.9 mmol/mol (-2.0 and 0.9% NGSP) at the 30 and 70 mmol/mol (4.9 and 8.6% NGSP) concentration level. Simulation of participation in the IFCC certification programme results in a Silver score (bias -0.1 mmol/mol, CV 1.1%). Interference in the presence of the most important Hb variants (AS, AC, AE, AD) and elevated HbA2 and HbF concentrations is less than 3 mmol/mol (0.3% NGSP) at a concentration of 50 mmol/mol (6.7% NGSP). CONCLUSIONS: Analytical performance of the HA-8190V is very good, especially with respect to precision and HbA1c quantification in the presence of the most common Hb variants.

Harmonization status of procalcitonin measurements: what do comparison studies and EQA schemes tell us?

Sepsis represents a global health priority because of its high mortality and morbidity. The key to improving prognosis remains an early diagnosis to initiate appropriate antibiotic treatment. Procalcitonin (PCT) is a recognized biomarker for the early indication of bacterial infections and a valuable tool to guide and individualize antibiotic treatment. To meet the increasing demand for PCT testing, numerous PCT immunoassays have been developed and commercialized, but results have been questioned. Many comparison studies have been carried out to evaluate analytical performance and comparability of results provided by the different commercially available immunoassays for PCT, but results are conflicting. External Quality Assessment Schemes (EQAS) for PCT constitute another way to evaluate results comparability. However, when making this comparison, it must be taken into account that the variety of EQA materials consist of different matrices, the commutability of which has not yet been investigated. The present study gathers results from all published comparison studies and results from 137 EQAS surveys to describe the current state-of-the-art harmonization of PCT results. Comparison studies globally highlight a significant variability of measurement results that nonetheless seem to have a moderate impact on medical decision-making. For their part, EQAS for PCT provides highly discrepant estimates of the interlaboratory CV. Due to differences in commutability of the EQA materials, the results from different peer groups could not be compared. To improve the informative value of the EQA data, the existing limitations such as non-harmonized conditions and suboptimal and/or unknown commutability of the EQA materials have to be overcome. The study highlights the need for commutable reference materials that could be used to properly evaluate result comparability and possibly standardize calibration, if necessary. Such an initiative would further improve the safe use of PCT in clinical routine.

IFCC Working Group Recommendations for Correction of Bias Caused by Noncommutability of a Certified Reference Material Used in the Calibration Hierarchy of an End-User Measurement Procedure.

Establishing metrological traceability to an assigned value of a matrix-based certified reference material (CRM) that has been validated to be commutable among available end-user measurement procedures (MPs) is central to producing equivalent results for the measurand in clinical samples (CSs) irrespective of the clinical laboratory MPs used. When a CRM is not commutable with CSs, the bias due to noncommutability will be propagated to the CS results causing incorrect metrological traceability to the CRM and nonequivalent CS results among different MPs. In a commutability assessment, a conclusion that a CRM is commutable or noncommutable for use with a specific MP is made when the difference in bias between the CRM and CSs meets or does not meet a criterion for that specific MP when compared to other MPs. A conclusion regarding commutability or noncommutability requires that the magnitude of the difference in bias observed in the commutability assessment remains unchanged over time. This conclusion requires the CRM to be stable and no substantive changes in the MPs. These conditions should be periodically reverified. If an available CRM is determined to be noncommutable for a specific MP, that CRM can be used in the calibration hierarchy for that MP when an appropriately validated MP-specific correction for the noncommutability bias is included. We describe with examples how a MP-specific correction and its uncertainty can be developed and applied in a calibration hierarchy to achieve metrological traceability of results for CSs to the CRM's assigned value.

Feasibility for aggregation of commutable external quality assessment results to evaluate metrological traceability and agreement among results.

Objectives: External quality assessment (EQA) with commutable samples is used for assessing agreement of results for patients' samples. We investigated the feasibility to aggregate results from four different EQA schemes to determine the bias between different measurement procedures and a reference target value. Methods: We aggregated EQA results for creatinine from programs that used commutable EQA material by calculating the relative difference between individual participant results and the reference target value for each sample. The means and standard errors of the means were calculated for the relative differences. Results were partitioned by methods, manufacturers and instrument platforms to evaluate the biases for the measurement procedures. Results: Data aggregated for enzymatic methods had biases that varied from -8.2 to 3.8% among seven instrument platforms for creatinine at normal concentrations (61-85 µmol/L). EQA schemes differed in the evidence provided about the commutability of their samples, and in the amount of detail collected from participants regarding the measurement procedures which limited the ability to sub-divide aggregated data by instrument platforms and models. Conclusions: EQA data could be aggregated from four different programs using different commutable samples to determine bias among different measurement procedures. Criteria for commutability for EQA samples as well as standardization of reporting the measurement methods, reagents, instrument platforms and models used by participants are needed to improve the ability to aggregate the results for optimal assessment of performance of measurement procedures. Aggregating data from a larger number of EQA schemes is feasible to assess trueness on a global scale.

Fast 0/1-h algorithm for detection of NSTEMI: are current high-sensitivity cardiac troponin assays fit for purpose? An EQA-based evaluation.

Background High-sensitivity cardiac troponin T/I (hs-cTnT/I) assays have improved analytical sensitivity for the detection of myocardial infarction (MI). To gain clinical specificity and sensitivity, interpretation of changes in cTn concentrations over time is crucial. The 2015 ESC NSTEMI guideline defines absolute delta values as additional rule-in and rule-out criteria for MI. A critical assumption for application of this rule is that total analytical imprecision within the delta period, including inter-instrument bias, is comparable to analytical imprecision in the validation studies. Methods Data from the Dutch External Quality Assessment Scheme (EQAS) were used to calculate inter-instrument bias and estimate imprecision for the measuring range where the proposed delta values are relevant: for Roche Elecsys hs-cTnT, 5-52 and 5-12 ng/L; for Abbott Architect hs-cTnI, 2-52 and 2-5 ng/L for rule-in and rule-out, respectively. Results For Elecsys, the median inter-instrument bias is 0.3 ng/L (n = 33 laboratories), resulting in reference change values (RCVs) of 3.0 and 1.7 ng/L, respectively, for rule-in and rule-out with imprecision as claimed by the manufacturer. With RCVs smaller than the guideline's delta thresholds, 100% of the laboratories have adequate specifications. RCVs for rule-in/rule-out increased to 4.6 ng/L/2.5 ng/L, respectively, with individual imprecisions as estimated from EQA data, resulting in 64% and 82% of laboratories with adequate specifications. For Architect, 40% of instruments (n = 10) might falsely qualify the result as clinically relevant; hence, inter-instrument bias could not be determined. Conclusions We advise laboratories that use the fast 0/1-h algorithm to introduce stringent internal quality procedures at the relevant/low concentration level, especially when multiple analyzers are randomly used.

Technical and clinical validation of the Greiner FC-Mix glycaemia tube.

BACKGROUND: Measurement of adequate glucose concentrations is complicated by in vitro breakdown of glucose due to glycolysis. Unlike the commonly used NaF-EDTA and NaF-oxalate phlebotomy tubes, citrated NaF-EDTA tubes are reported to directly and thereby completely inhibit glycolysis. Recently, Greiner introduced the Vacuette® FC-Mix NaF-EDTA-citrate tube, currently the only NaF-citrate tube without volume-disturbing liquid additions available on the European market. Here we present its potential as alternative for the laborious and therefore unfeasible conditions for glucose sampling as recommended by the World Health Organization (WHO). METHODS: The FC-Mix tube was tested against the WHO recommended method of optimal laboratory conditions, both in healthy volunteers and pregnant woman undergoing oral glucose tolerance test (oGTT) for screening of gestational diabetes mellitus (GDM). Glucose concentrations were measured after different incubation times (0-48 h) and temperatures (room temperature, 37 °C), both in uncentrifuged whole blood and centrifuged material. RESULTS: Deming regression analysis shows that glucose concentrations measured in the FC-Mix tube correlate to the WHO recommended method. Stability is maintained at room temperature for 48 h and at least 24 h at 37 °C. The use of the FC-Mix tube was also validated in screening for GDM and proved comparable to the WHO recommended method in diagnostic outcome. CONCLUSIONS: The new Greiner FC-Mix tube combines the easy handling of a routine tube with dry additive with the ability to immediately inhibit glycolysis as in the WHO method for optimal pre-analytical and analytical conditions and performs equally to those conditions when screening for GDM.

ß1-Adrenergic receptor signaling activates the epithelial calcium channel, transient receptor potential vanilloid type 5 (TRPV5), via the protein kinase A pathway.

Epinephrine and norepinephrine are present in the pro-urine. ß-Adrenergic receptor (ß-AR) blockers administered to counteract sympathetic overstimulation in patients with congestive heart failure have a negative inotropic effect, resulting in reduced cardiac contractility. Positive inotropes, ß1-AR agonists, are used to improve cardiac functions. Active Ca(2+) reabsorption in the late distal convoluted and connecting tubules (DCT2/CNT) is initiated by Ca(2+) influx through the transient receptor potential vanilloid type 5 (TRPV5) Ca(2+) channel. Although it was reported that ß-ARs are present in the DCT2/CNT region, their role in active Ca(2+) reabsorption remains elusive. Here we revealed that ß1-AR, but not ß2-AR, is localized with TRPV5 in DCT2/CNT. Subsequently, treatment of TRPV5-expressing mouse DCT2/CNT primary cell cultures with the ß1-AR agonist dobutamine showed enhanced apical-to-basolateral transepithelial Ca(2+) transport. In human embryonic kidney (HEK293) cells, dobutamine was shown to stimulate cAMP production, signifying functional ß1-AR expression. Fura-2 experiments demonstrated increased activity of TRPV5 in response to dobutamine, which could be prevented by the PKA inhibitor H89. Moreover, nonphosphorylable T709A-TRPV5 and phosphorylation-mimicking T709D-TRPV5 mutants were unresponsive to dobutamine. Surface biotinylation showed that dobutamine did not affect plasma membrane abundance of TRPV5. In conclusion, activation of ß1-AR stimulates active Ca(2+) reabsorption in DCT2/CNT; an increase in TRPV5 activity via PKA phosphorylation of residue Thr-709 possibly plays an important role. These data explicate a calciotropic role in addition to the inotropic property of ß1-AR.

Shedding of klotho by ADAMs in the kidney.

The anti-aging gene klotho plays an important role in Ca(2+) and phosphate homeostasis. Membrane-bound klotho is an essential coreceptor for fibroblast growth factor-23 and can be cleaved by proteases, including a disintegrin and metalloproteinase (ADAM)10 and ADAM17. Cleavage of klotho occurs at a site directly above the plasma membrane (α-cut) or between the KL1 and KL2 domain (ß-cut), resulting in soluble full-length klotho or KL1 and KL2 fragments, respectively. The aim of the present study was to gain insights into the mechanisms behind klotho cleavage processes in the kidney. Klotho shedding was demonstrated using a Madin-Darby canine kidney cell line stably expressing klotho and human embryonic kidney-293 cells transiently transfected with klotho. Here, we report klotho expression on both the basolateral and apical membrane, with a higher abundance of klotho at the apical membrane and in the apical media. mRNA expression of ADAM17 and klotho were enriched in mouse distal convoluted and connecting tubules. In vitro ADAM/matrix metalloproteinase inhibition by TNF484 resulted in a concentration-dependent inhibition of the α-cut, with a less specific effect on ß-cut shedding. In vivo TNF484 treatment in wild-type mice did not change urinary klotho levels. However, ADAM/matrix metalloproteinase inhibition did increase renal and duodenal mRNA expression of phosphate transporters, whereas serum phosphate levels were significantly decreased. In conclusion, our data show that renal cells preferentially secrete klotho to the apical side and suggest that ADAMs are responsible for α-cut cleavage.

Coordinated regulation of TRPV5-mediated Ca²âº transport in primary distal convolution cultures.

Fine-tuning of renal calcium ion (Ca(2+)) reabsorption takes place in the distal convoluted and connecting tubules (distal convolution) of the kidney via transcellular Ca(2+) transport, a process controlled by the epithelial Ca(2+) channel Transient Receptor Potential Vanilloid 5 (TRPV5). Studies to delineate the molecular mechanism of transcellular Ca(2+) transport are seriously hampered by the lack of a suitable cell model. The present study describes the establishment and validation of a primary murine cell model of the distal convolution. Viable kidney tubules were isolated from mice expressing enhanced Green Fluorescent Protein (eGFP) under the control of a TRPV5 promoter (pTRPV5-eGFP), using Complex Object Parametric Analyser and Sorting (COPAS) technology. Tubules were grown into tight monolayers on semi-permeable supports. Radioactive (45)Ca(2+) assays showed apical-to-basolateral transport rates of 13.5 ± 1.2 nmol/h/cm(2), which were enhanced by the calciotropic hormones parathyroid hormone and 1,25-dihydroxy vitamin D3. Cell cultures lacking TRPV5, generated by crossbreeding pTRPV5-eGFP with TRPV5 knockout mice (TRPV5(-/-)), showed significantly reduced transepithelial Ca(2+) transport (26 % of control), for the first time directly confirming the key role of TRPV5. Most importantly, using this cell model, a novel molecular player in transepithelial Ca(2+) transport was identified: mRNA analysis revealed that ATP-dependent Ca(2+)-ATPase 4 (PMCA4) instead of PMCA1 was enriched in isolated tubules and downregulated in TRPV5(-/-) material. Immunohistochemical stainings confirmed co-localization of PMCA4 with TRPV5 in the distal convolution. In conclusion, a novel primary cell model with TRPV5-dependent Ca(2+) transport characteristics was successfully established, enabling comprehensive studies of transcellular Ca(2+) transport.

Identification of the human zinc transcriptional regulatory element (ZTRE): a palindromic protein-binding DNA sequence responsible for zinc-induced transcriptional repression.

Many genes with crucial roles in zinc homeostasis in mammals respond to fluctuating zinc supply through unknown mechanisms, and uncovering these mechanisms is essential to understanding the process at cellular and systemic levels. We detected zinc-dependent binding of a zinc-induced protein to a specific sequence, the zinc transcriptional regulatory element (ZTRE), in the SLC30A5 (zinc transporter ZnT5) promoter and showed that substitution of the ZTRE abrogated the repression of a reporter gene in response to zinc. We identified the ZTRE in other genes, including (through an unbiased search) the CBWD genes and (through targeted analysis) in multiple members of the SLC30 family, including SLC30A10, which is repressed by zinc. The function of the CBWD genes is currently unknown, but roles for homologs in metal homeostasis are being uncovered in bacteria. We demonstrated that CBWD genes are repressed by zinc and that substitution of the ZTRE in SLC30A10 and CBWD promoter-reporter constructs abrogates this response. Other metals did not affect expression of the transcriptional regulator, binding to the ZTRE or promoter-driven reporter gene expression. These findings provide the basis for elucidating how regulation of a network of genes through this novel mechanism contributes to zinc homeostasis and how the cell orchestrates this response.

Role of the transient receptor potential vanilloid 5 (TRPV5) protein N terminus in channel activity, tetramerization, and trafficking.

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry site for active Ca(2+) reabsorption in the kidney. The TRPV5 channel is a member of the TRP family of cation channels, which are composed of four subunits together forming a central pore. Regulation of channel activity is tightly controlled by the intracellular N and C termini. The TRPV5 C terminus regulates channel activity by various mechanisms, but knowledge regarding the role of the N terminus remains scarce. To study the role of the N terminus in TRPV5 regulation, we generated different N-terminal deletion constructs. We found that deletion of the first 32 residues did not affect TRPV5-mediated (45)Ca(2+) uptake, whereas deletion up to residue 34 and 75 abolished channel function. Immunocytochemistry demonstrated that these mutant channels were retained in the endoplasmic reticulum and in contrast to wild-type TRPV5 did not reach the Golgi apparatus, explaining the lack of complex glycosylation of the mutants. A limited amount of mutant channels escaped the endoplasmic reticulum and reached the plasma membrane, as shown by cell surface biotinylation. These channels did not internalize, explaining the reduced but significant amount of these mutant channels at the plasma membrane. Wild-type TRPV5 channels, despite significant plasma membrane internalization, showed higher plasma membrane levels compared with the mutant channels. The assembly into tetramers was not affected by the N-terminal deletions. Thus, the N-terminal residues 34-75 are critical in the formation of a functional TRPV5 channel because the deletion mutants were present at the plasma membrane as tetramers, but lacked channel activity.