Pectoralis and Serratus Fascial Plane Blocks Each Provide Early Analgesic Benefits Following Ambulatory Breast Cancer Surgery: A Retrospective Propensity-Matched Cohort Study.

BACKGROUND: Pectoralis and serratus blocks have been described recently for use in breast surgery, but evidence supporting their analgesic benefits is limited. This cohort study evaluates the benefits of adding a pectoralis or serratus block to conventional opioid-based analgesia (control) in patients who underwent ambulatory breast cancer surgery at Women's College Hospital between July 2013 and May 2015. We tested the joint hypothesis that adding a pectoralis or serratus block reduced postoperative in-hospital (predischarge) opioid consumption and nausea and vomiting (PONV). We also examined the 2 block types for noninferiority. METHODS: A total of 225 patients were propensity matched on 5 potential confounders among 3 study groups (75 per group): (1) pectoralis; (2) serratus; and (3) control. The propensity-matched cohort was used to evaluate the effect of the study group on postoperative in-hospital oral morphine equivalent consumption and PONV. We considered pectoralis noninferior to serratus block if it was noninferior for both outcomes, within 10 mg morphine and 17.5% in PONV incidence margins. Other outcomes included intraoperative fentanyl requirements, pain scores, time to first analgesic request, and duration of recovery room stay. RESULTS: Both pectoralis and serratus blocks were each associated with reduced postoperative in-hospital opioid consumption and PONV compared with control. Pectoralis was noninferior to serratus block for these 2 outcomes. Pectoralis and serratus blocks were each associated with reduced intraoperative fentanyl requirements, prolonged time to first analgesic request, and expedited recovery room discharge compared with control; there were no differences for the remaining outcomes. CONCLUSIONS: Pectoralis and serratus blocks were each associated with a reduction in postoperative in-hospital opioid consumption and PONV compared with conventional opioid-based analgesia after ambulatory breast cancer surgery.

Is sciatic nerve block advantageous when combined with femoral nerve block for postoperative analgesia following total knee arthroplasty? a meta-analysis.

BACKGROUND: Total knee arthroplasty (TKA) is associated with moderate-to-severe postoperative pain despite the use of femoral nerve block (FNB). The analgesic benefits of adding sciatic nerve block (SNB) to FNB following TKA are unclear. The aim of this meta-analysis was to quantify the analgesic effects of adding SNB to FNB following TKA. METHODS: We searched the US National Library of Medicine (MEDLINE), Excerpta Medica (Embase), and Cochrane Central Controlled Trials Register databases in March 2015 for randomized and quasi-randomized controlled trials (RCTs) that evaluated the analgesic advantages of adding SNB to FNB compared to FNB alone after TKA. The designated primary outcome was intravenous morphine consumption during the 24-hr postoperative interval. The severity of pain was evaluated at rest and with movement two, four, eight, 12, 24, 36, and 48 hr postoperatively. Morphine consumption during the postoperative 24-48 hr interval, time to first analgesic request, opioid-related side effects, block-related complications, patient satisfaction, functional recovery, and time to hospital discharge were also evaluated. Trials were stratified based on whether a single-shot SNB (SSNB) or continuous SNB (CSNB) was used. Data were combined using random effects modelling. RESULTS: Eight RCTs, including 379 patients, were analyzed. Five trials examined SSNB, and three assessed CSNB. Together, SSNB and CSNB reduced the 0-24 hr weighted mean difference [95% confidence interval] of morphine consumption by 10.6 [-20.9 to -0.3] mg (P = 0.042; I(2) = 97%) and 20.5 [-28.6 to -12.4] mg (P < 0.001, I(2) = 86%), respectively. SSNB reduced pain at rest and during movement up to 8 hr postoperatively (P = 0.023 and P < 0.001, respectively), whereas CSNB reduced pain at rest up to 36 hr (P = 0.004) and pain with movement up to 48 hr (P = 0.031). CSNB also decreased the odds of postoperative nausea and vomiting by 91% (P = 0.011). CONCLUSION: The available evidence supporting the analgesic benefits of adding SNB to FNB following TKA is marked by significant heterogeneity. With this challenge in mind, our meta-analysis suggests that SNB can significantly reduce postoperative opioid consumption and diminish knee pain following TKA compared to no SNB in the setting of FNB.

Ultrasound guided distal peripheral nerve block of the upper limb: A technical review.

Upper extremity surgery is commonly performed under regional anesthesia. The advent of ultrasonography has made performing upper extremity nerve blocks relatively easy with a high degree of reliability. The proximal approaches to brachial plexus block such as supraclavicular plexus block, infraclavicular plexus block, or the axillary block are favored for the most surgical procedures of distal upper extremity. Ultrasound guidance has however made distal nerve blocks of the upper limb a technically feasible, safe and efficacious option. In recent years, there has thus been a resurgence of distal peripheral nerve blocks to facilitate hand and wrist surgery. In this article, we review the technical aspects of performing the distal blocks of the upper extremity and highlight some of the clinical aspects of their usage.

Effect of rapid fluid resuscitation using crystalloids or colloids on hemostasis in piglets.

BACKGROUND AND OBJECTIVES: Impairment of blood coagulation is one of the main side effects of volume replacement, particularly if artificial colloids such as hydroxyethyl starch (HES) and gelatine preparations are used. This animal study aimed to evaluate the effect of a single fast intravenous crystalloid or colloid fluid bolus on blood coagulation as measured by rotation thromboelastometry (ROTEM). METHODS: Thirty-two anesthetized piglets were infused with a rapid 20 ml·kg(-1) fluid bolus of either normal saline (NS), 4% gelatine, 5% albumin or 6% HES 130/0.4 (n = 8 per group) over a period of 2 min. Hemostasis was assessed by ROTEM before and 1 min after fluid administration. Within-group differences were analyzed by Wilcoxon test, and additionally overall Kruskal-Wallis test followed by posthoc Mann-Whitney U-test were applied to detect differences between groups. RESULTS: All fluids caused a significant weakening of clot strength within groups. HES and gelatine showed a significantly stronger impairment of clot growth and maximum clot firmness as compared with albumin and normal saline. Impairment of fibrin polymerization was more pronounced following HES as compared with all other fluids. CONCLUSION: After moderate but very fast volume loading, HES and gelatine impair blood coagulation to a larger extent as compared with albumin or normal saline, while no significant differences were observed between both artificial colloids.

Resuscitation and transfusion management in trauma patients: emerging concepts.

PURPOSE OF REVIEW: Severe trauma is associated with hemorrhage, coagulopathy and transfusion of blood and blood products, all associated with considerable mortality and morbidity. The aim of this review is to focus on resuscitation, transfusion strategies and the management of bleeding in trauma as well as to emphasize on why coagulation has to be monitored closely and to discuss the rationale of modern and future transfusion strategies. RECENT FINDINGS: Coagulopathy and uncontrolled bleeding remain leading causes of death in trauma, lead to blood transfusions and increased mortality as it has been recently shown that blood transfusion per se results in an adverse outcome. In the last years, damage control resuscitation, a combination of permissive hypotension, hemostatic resuscitation and damage control surgery, has been introduced to treat severely traumatized patients in hemorrhagic shock. Goals of treatment in trauma patients remain avoiding metabolic acidosis, hypothermia, treating coagulopathy and stabilizing the patient as soon as possible. The place of colloids and crystalloids in trauma resuscitation as well as the role of massive transfusion protocols with a certain FFP : RBC ratio and even platelets have to be reevaluated. SUMMARY: Close monitoring of bleeding and coagulation in trauma patients allows goal-directed transfusions and thereby optimizes the patient's coagulation, reduces the exposure to blood products, reduces costs and may improve clinical outcome.

Perioperative course of FXIII in children undergoing major surgery.

BACKGROUND: Acquired deficiency of FXIII because of perioperative hemodilution has been described several times in adults; however, data in children are scarce. We performed a prospective observational trial to evaluate the intraoperative course of FXIII in children undergoing elective major surgery. METHODS: Blood samples were repeatedly taken from 46 children aged 0.3-16 years undergoing major surgery. Concentrations of FXIII and fibrinogen, thrombelastometry by ROTEM®, and cell count were assessed intraoperatively. RESULTS: A significant decrease in FXIII concentration (median 60%; IQR 49-69%) was already noted at beginning of surgical procedures, while most ROTEM® traces remain unchanged. FXIII levels further deteriorated intraoperatively to minimal levels of 33% (15-61%). Lowest intraoperative clot strength (ExTEM) was 44 mm (34-50 mm), and fibrinogen plasma levels decreased to minimal levels of 130 mg·dl(-1) (95-160 mg·dl(-1) ). In 43 of 46 children, transfusion therapy was necessary. Despite of transfusion of fresh frozen plasma (cumulative total dose 22 ml·kg(-1) [11-32 ml·kg(-1) ]) in 21 of 46 children, FXIII level remains low in all children till the end of surgery at levels of 39% (20-46%). CONCLUSIONS: Coagulation factor XIII decreased early during major surgery owing to hemodilution. Overall intraoperative FXIII levels remain low despite of transfusion of fresh frozen plasma.

Comparison of point-of-care testing (POCT): i-STAT(®) international normalized ratio (INR) vs reference laboratory INR in pediatric patients undergoing major surgery.

BACKGROUND: The aim of the study was to compare international normalized ratio (INR) results obtained by point-of-care testing (i-STAT® device) with the reference laboratory INR in children undergoing major surgery with expected significant blood loss. METHODS: Pediatric patients undergoing craniofacial, spine, hip, or cancer surgery were included. Blood samples for coagulation testing were tested at several intraoperative time points and generally withdrawn from the arterial catheter, if accessible. A volume of 1.4 ml citrated blood was used for the reference laboratory INR test, and 0.1 ml of blood was taken for the whole blood INR test using the i-STAT® device. Blood samples for both tests were withdrawn at the same time and immediately analyzed with both devices. RESULTS: A total of 169 paired blood samples were taken intraoperatively from 44 pediatric patients [IQR 0.9-10.7 years (median 3.3)]. Reference laboratory INR ranged from 0.96 to 3.43 (mean 1.40; sd 0.32) and INR of i-STAT® from 0.95 to 2.29 (mean 1.26; sd 0.22). The correlation coefficient was 0.83 (P < 0.001), and the bias values were 0.12 and 0.55 at the medical decision level of ≤2.0 and >2.0, respectively. CONCLUSIONS: In the perioperative setting, point-of-care INR testing in children using the i-STAT® device is a reliable and easy-to-handle method for INR values ≤2.0, while INR values >2.0 might be underestimated.

Impact of propofol on electrocardiographic alterations during intravascular application of bupivacaine--a study in piglets.

BACKGROUND: Intravascular application of a small dose of local anesthetics (LA) with epinephrine as well as larger doses of LA under sevoflurane anesthesia results in increase in T-wave amplitude in the electrocardiogram (ECG). The aim of this study was to elucidate whether propofol anesthesia affects these ECG alterations or not. METHODS: Thirty neonatal pigs were randomized into two groups. Group 1 was anesthetized with sevoflurane, group 2 with sevoflurane plus continuous propofol infusion (10 mg·kg(-1)·h(-1)). A test dose of 0.2 ml·kg(-1) bupivacaine 0.125% + epinephrine 1 : 200,000 was injected intravenously. Arterial pressure was monitored. ECG was analyzed for changes in T-wave amplitude (positive if ≥25% baseline) and heart rate. In another setting, bupivacaine 0.125% was intravenous infused at a rate of 4 mg·kg(-1)·min(-1). ECG was analyzed for alteration in T-wave amplitude and heart rate at 1.25, 2.5, and 5 mg·kg(-1) bupivacaine infused. RESULTS: T-wave elevation after the administration of an epinephrine containing LA test dose was similar between the two groups. Increase in heart rate caused by the test dose were significantly higher in group 2 (P = 0.008). During continuous bupivacaine administration, T-wave elevation occurred in 40% and 71% (group 1 and 2) at 1.25 mg·kg(-1), in 80% and 100% at 2.5 mg·kg(-1), and in 93% and 86% at 5 mg·kg(-1) bupivacaine infused. CONCLUSION: Continuous propofol infusion does not suppress the ECG signs of a systemically administered epinephrine containing LA test dose nor does it suppress the ECG signs caused by high doses of intravenous applied bupivacaine.

Tracheal fluid leakage in benchtop trials: comparison of static versus dynamic ventilation model with and without lubrication.

PURPOSE: Longitudinal folds in tracheal tube (TT) cuffs cause leakage of pooled secretions past the tube cuff, and the most common in vitro method to test the efficacy of a new tube is a benchtop model using an artificial rigid trachea. This study compared the potential of a static and dynamic ventilation benchtop model and cuff lubrication in testing the tracheal sealing properties of a given TT cuff. METHODS: Static trial Six brands of 7.5 mm internal diameter (ID) cuffed TT (n = 8) with high volume-low pressure cuffs were inflated in an artificial trachea (18 mm ID) without and with lubrication. Dynamic trial The same tube cuffs, without lubrication, were subjected to positive pressure ventilation (PPV) + positive end-expiratory pressure (PEEP) of 5cmH(2)O or to PPV alone (without PEEP) or to PEEP alone (without PPV). Clear water (5 ml) was placed above the tube cuff, and fluid leakage (ml) was measured up to 60 min. RESULTS: Gel lubrication, PEEP alone and PPV + PEEP completely prevented fluid leakage across the tube cuffs in all six TT brands tested within 60 min when compared to the static unlubricated model (0% leak versus 100% leak; P < 0.01). Fluid leakage in the static unlubricated model and the PPV group was 1.38-4.76 ml and 0.23-4.47 ml, respectively. CONCLUSION: Gel lubrication, PEEP alone, and PPV + PEEP in the benchtop model had a much stronger protective effect than PPV alone on fluid leakage. Studies testing the fluid sealing efficiency of tube cuffs might be more conclusive in a static benchtop model without lubrication than in a dynamic model.

Too Deep or Not Too Deep?: A Propensity-Matched Comparison of the Analgesic Effects of a Superficial Versus Deep Serratus Fascial Plane Block for Ambulatory Breast Cancer Surgery.

BACKGROUND AND OBJECTIVES: Serratus fascial plane block can reduce pain following breast surgery, but the question of whether to inject the local anesthetic superficial or deep to the serratus muscle has not been answered. This cohort study compares the analgesic benefits of superficial versus deep serratus plane blocks in ambulatory breast cancer surgery patients at Women's College Hospital between February 2014 and December 2016. We tested the joint hypothesis that deep serratus block is noninferior to superficial serratus block for postoperative in-hospital (pre-discharge) opioid consumption and pain severity. METHODS: One hundred sixty-six patients were propensity matched among 2 groups (83/group): superficial and deep serratus blocks. The cohort was used to evaluate the effect of blocks on postoperative oral morphine equivalent consumption and area under the curve for rest pain scores. We considered deep serratus block to be noninferior to superficial serratus block if it were noninferior for both outcomes, within 15 mg morphine and 4 cm·h units margins. Other outcomes included intraoperative fentanyl requirements, time to first analgesic request, recovery room stay, and incidence of postoperative nausea and vomiting. RESULTS: Deep serratus block was associated with postoperative morphine consumption and pain scores area under the curve that were noninferior to those of the superficial serratus block. Intraoperative fentanyl requirements, time to first analgesic request, recovery room stay, and postoperative nausea and vomiting were not different between blocks. CONCLUSIONS: The postoperative in-hospital analgesia associated with deep serratus block is as effective (within an acceptable margin) as superficial serratus block following ambulatory breast cancer surgery. These new findings are important to inform both current clinical practices and future prospective studies.

Expression of visinin-like protein-3 in mouse kidney.

In renal proximal brush borders the Na/Pi cotransporter NaPi-IIa is part of a heteromultimeric complex including the PDZ proteins PDZK1 and NHERF1, which interact with the C terminus of NaPi-IIa. In this study, a yeast two-hybrid screen against the N terminus of the Na/Pi cotransporter NaPi-IIa was performed. Thereby we identified visinin-like protein-3 (VILIP-3), a member of neuronal calcium sensors. In this study, expression and protein localization of VILIP-3 in the mouse kidney was performed by immunofluorescence and RT-PCR using laser-assisted microdissected nephron segments. VILIP-3 was found to be abundant in distal and collecting ducts where it partly colocalized with calbindin D28K. In addition VILIP-3 was observed in the brush borders of proximal tubular S1 and S3 segments of both superficial and deep nephrons.

Massive aspiration past the tracheal tube cuff caused by closed tracheal suction system.

BACKGROUND: Aspiration past the tracheal tube cuff has been recognized to be a risk factor for the development of ventilator-associated pneumonia (VAP). This study investigated the effect of closed tracheal suctioning on aspiration of fluid past the tracheal tube cuff in an in vitro benchtop model. METHODS: High-volume low pressure tube cuffs of 7.5 mm internal diameter (ID) were placed in a 22 mm ID artificial trachea connected to a test lung. Positive pressure ventilation (PPV) with 15 cm H2O peak inspiratory pressure and 5 cm H2O positive end-expiratory pressure (PEEP) was used. A closed tracheal suction system (CTSS) catheter (size 14Fr) was attached to the tracheal tube and suction was performed for 5, 10, 15, or 20 seconds under 200 or 300 cm H2O suction pressures. Amount of fluid (mL) aspirated along the tube cuff and the airway pressure changes were recorded for each suction procedure. Fluid aspiration during different suction conditions was compared using Kruskal-Wallis and Mann-Whitney test (Bonferroni correction [α = .01]). RESULTS: During 10, 15, and 20 seconds suction, airway pressure consistently dropped down to -8 to -13 cm H2O (P < .001) from the preset level. Fluid aspiration was never observed under PPV + PEEP but occurred always during suctioning. Aspiration along the tube cuff was higher with -300 cm H2O than with -200 cm H2O suction pressure (P < .001) and was much more during 15 and 20 seconds suction time as compared to 5 seconds (P < .001). CONCLUSION: Massive aspiration of fluid occurs along the tracheal tube cuff during suction with the closed tracheal suction system.

Intrarater and interrater variability of point of care coagulation testing using the ROTEM delta.

This study aimed to assess intrarater and interrater variability of coagulation point-of-care testing (POCT) using ROTEM delta operated by trained staff. Arterial blood samples were taken from 43 anesthetized piglets aged up to 6 weeks and weighing 4-6 kg. The following clotting measurements were recorded: clotting time, clot formation time (CFT), maximum clot firmness (MCF) and alpha angle using ROTEM delta assays ExTEM, InTEM, FibTEM and ApTEM. Intrarater variability was assessed when a single operator performed the same assay simultaneously in all four channels of the ROTEM device. Interrater variability was assessed by two different operators simultaneously performing the same assay. Variance components of the data were analyzed using linear mixed modeling. Three hundred and forty-three tests from 86 samples were loaded and analyzed. The intraclass correlation coefficient (ICC) was more than 0.7 for clotting measurements except for CFT and alpha in InTEM. For intrarater and interrater assessment, different relative variability for the ROTEM measurements were found with consistently higher variability for clotting time and CFT and lower variability of MCF and alpha angle. Interrater variability was not statistically significant as supported using Akaike's information criterion. Piglet coagulation testing using ROTEM delta showed a high ICC. Variability was significantly lower in MCF and angle alpha compared with clotting time and CFT. No further variability was added by a second user. Based on these data, ROTEM delta appears to be suitable as POCT.

Allogeneic red blood cell transfusion: physiology of oxygen transport.

Allogeneic red blood cell (RBC) transfusions have been shown to be associated with considerable risks. While their efficiency in many clinical situations has not been proven, the number of studies finding adverse outcomes in terms of morbidity (e.g. postoperative infections) and mortality continues to rise. In view of these facts, physicians involved in transfusion medicine have to be as restrictive as possible with RBC transfusions. Only a thorough knowledge of the physiology and pathophysiology of oxygen transport can be a solid base for meaningful transfusion decisions. Therefore, the goal of this article is to review the basics of oxygen transport and normovolaemic anaemia.

Novel starches: single-dose pharmacokinetics and effects on blood coagulation.

BACKGROUND: Carboxymethyl starch (CMS) and carboxymethylated hydroxyethyl starch (CM-HES) might offer advantages over hydroxyethyl starch (HES) with regard to their volume expansion effect and their pharmacokinetic characteristics. The goal of the current study was to determine the pharmacokinetics of CMS and CM-HES and to investigate their influence on blood coagulation in comparison with the standard low-molecular, low-substituted HES (130/0.42) used in Europe. METHODS: The study was conducted as a randomized, blinded, parallel three-group study in 30 pigs. Twenty ml/kg of 6% HES (control), 6% CMS, or 6% CM-HES was infused as a single dose, and serial blood sampling was performed over 20 h to measure plasma concentration and molecular weight and to assess blood coagulation. Concentration-effect relations were assessed by pharmacokinetic-pharmacodynamic analysis. RESULTS: CMS and CM-HES showed significantly higher plasma concentrations and molecular weights over 20 h (P for both<0.001) with smaller volumes of distribution and longer elimination rates during the terminal phase (P for both<0.01) when compared with HES. CMS and CM-HES impaired whole blood coagulation more than HES as assessed by Thrombelastograph analysis (Haemoscope Corporation, Niles, IL). However, similar effects of all three starch preparations on blood coagulation were found when related to the plasma concentrations in mass units. CONCLUSIONS: Carboxymethylation of starch results in an increased intravascular persistence and a slower fragmentation compared with HES. The greater impairment of blood coagulation by CMS and CM-HES seems to be caused by the higher plasma concentrations.

Anemia and perioperative red blood cell transfusion: a matter of tolerance.

In the past, anemia in the perioperative period has been treated by red blood cell (RBC) transfusions relatively uncritically. RBC transfusions were believed to increase oxygen delivery by increasing hemoglobin concentration. Arbitrary transfusion triggers such as the "10/30 rule" (i.e., RBC transfusion indicated below a hemoglobin concentration of 10 g/dL or a hematocrit of 30%) were applied. However, there is now increasing evidence that RBC transfusions are associated with adverse outcomes and should be avoided whenever possible. Restraining from RBC transfusions and maintaining normovolemia in patients suffering from surgical blood loss results in acute anemia. Therefore, knowing the compensatory mechanisms during acute anemia is crucial. This review focuses on acute anemia tolerance, its limits, and physiologic transfusion triggers in the perioperative period.

Effect of high- and low-molecular-weight low-substituted hydroxyethyl starch on blood coagulation during acute normovolemic hemodilution in pigs.

BACKGROUND: Hydroxyethyl starches (HES) with lower impact on blood coagulation but longer intravascular persistence are of clinical interest. The current study aimed to investigate in vivo the isolated effect of molecular weight on blood coagulation during progressive acute normovolemic hemodilution. METHODS: Twenty-four pigs were normovolemically hemodiluted up to a total exchange of 50 ml . kg . body weight of HES 650/0.42 or HES 130/0.42. Serial blood sampling was performed to measure HES plasma concentration and to assess blood coagulation. Concentration-effect relations were analyzed by linear regression, followed by the Student t test on regression parameters. RESULTS: Blood coagulation was increasingly compromised toward hypocoagulability by acute normovolemic hemodilution with both treatments (P < 0.01). Significantly greater impact on activated partial thromboplastin time (P = 0.04) and significantly stronger decrease of maximal amplitude (P = 0.04), angle alpha (P = 0.02), and coagulation index (P = 0.02) was seen after acute normovolemic hemodilution with HES 650/0.42 as compared with HES 130/0.42. Except for factor VIII (P = 0.04), no significant differences between both treatments were observed when relating antihemostatic effects to HES plasma concentrations (P > 0.05). A significantly lesser decrease of hemoglobin concentration has been found with HES 650/0.42 as compared with HES 130/0.42 (P < 0.01) in relation to HES plasma concentrations. CONCLUSION: High-molecular-weight HES (650/0.42) shows a moderately greater antihemostatic effect than low-molecular-weight HES (130/0.42) during acute normovolemic hemodilution. However, similar effects on hemostasis were observed with both treatments when observed antihemostatic effects were related to measured HES plasma concentrations. In addition, HES 650/0.42 may have a lower efficacy in immediately restoring plasma volume.

Allogeneic blood transfusions: benefit, risks and clinical indications in countries with a low or high human development index.

The risks associated with allogeneic red blood cell (RBC) transfusions differ significantly between countries with low and high human development indexes (HDIs). In countries with a low HDI, the risk of infection (HIV, HBV, HCV and malaria) is elevated. In contrast, in countries with a high HDI, immunological reactions (haemolytic transfusion reactions, alloimmunization and immunosuppression) are predominant. Therefore the overall risk associated with RBC transfusions in low HDI countries is much more significant than that in high HDI countries. In view of these risks, the limited efficacy of RBC transfusion and its high costs, this procedure should be used sparingly and rationally. Therefore RBC transfusion protocols adapted to the local situation are essential. Such protocols should distinguish between physiological and haemoglobin-based transfusion triggers. In countries with a high HDI, relative tachycardia and hypotension, despite normovolaemia, ST-segment changes suggestive of myocardial ischaemia and an Hb level <6 g/dl can serve as general guidelines for transfusion. Higher haemoglobin transfusion triggers should be used for patients aged >80 years and those with coronary artery or cerebrovascular disease. In countries with a low HDI, clinical signs of circulatory failure or myocardial ischaemia and an Hb level <5 g/dl can serve as transfusion guidelines.

PDZK1: II. an anchoring site for the PKA-binding protein D-AKAP2 in renal proximal tubular cells.

BACKGROUND: PDZK1, a multiple PDZ protein, was recently found to interact with the type IIa Na/Pi cotransporter (NaPi-IIa) in renal proximal tubular cells. In a preceding study, yeast two-hybrid screens using single PDZ domains of PDZK1 as baits were performed. Among the identified proteins, a C-terminal fragment of the dual-specific A-kinase anchoring protein 2 (D-AKAP2) was obtained by screening PDZ domain 4. METHODS: After its molecular cloning by means of RACE, the renal expression of D-AKAP2 was analyzed by real-time polymerase chain reaction (PCR) and immunohistochemistry. Protein interactions were characterized by overlays, pull-downs, and immunoprecipitations from transfected opossum kidney (OK) cells. RESULTS: Based on 5'-RACE and PDZK1 overlays of mouse kidney cortex separated by two-dimensional electrophoresis, it was suggested that the renal isoform of D-AKAP2 in mouse comprises 372 amino acids and exists as a protein of >40 kD. Immunohistochemistry and real-time PCR localized D-AKAP2 only to the subapical pole of proximal tubular cells in mouse kidney. In pull-down experiments, D-AKAP2 tightly bound PDZK1 as well as N+/H+ exchanger regulator factor (NHERF-1), but the latter with an apparent fourfold lower affinity. Similarly, His-tagged D-AKAP2 specifically retained PDZK1 from mouse kidney cortex homogenate. In addition, myc-tagged D-AKAP2 and HA-tagged PDZK1 co-immunoprecipitated from transfected OK cells. CONCLUSION: We conclude that D-AKAP2 anchors protein kinase A (PKA) to PDZK1 and to a lesser extent to NHERF-1. Since PDZK1 and NHERF-1 both sequester NaPi-IIa to the apical membrane, D-AKAP2 may play an important role in the parathyroid hormone (PTH)-mediated regulation of NaPi-IIa by compartmentalization of PKA.

Interactions of MAP17 with the NaPi-IIa/PDZK1 protein complex in renal proximal tubular cells.

An essential role in phosphate homeostasis is played by Na/Pi cotransporter IIa that is localized in the brush borders of renal proximal tubular cells. Recent studies identified several PDZ proteins interacting with the COOH-terminal tail of NaPi-IIa, such as PDZK1 and NHERF-1. Here, by using yeast two-hybrid screen of mouse kidney cDNA library, we attempted to find proteins interacting with the NH2-terminal part of NaPi-IIa. We identified MAP17, a 17-kDa membrane protein that has been described to be associated with various human carcinomas, but it is also expressed in normal kidneys. Results obtained by various in vitro analyses suggested that MAP17 interacts with the fourth domain of PDZK1 but not with other PDZ proteins localized in proximal tubular brush borders. As revealed by immunofluorescence, MAP17 was abundant in S1 but almost absent in S3 segments. No alterations of the apical abundance of MAP17 were observed after maneuvers undertaken to change the content of NaPi-IIa (parathyroid hormone treatment, different phosphate diets). In agreement, no change in the amount of MAP17 mRNA was observed. Results obtained from transfection studies using opossum kidney cells indicated that the apical localization of MAP17 is independent of PDZK1 but that MAP17 is required for apical localization of PDZK1. In summary, we conclude that MAP17 1) interacts with PDZK1 only, 2) associates with the NH2 terminus of NaPi-IIa within the PDZK1/NaPi-IIa/MAP17 complex, and 3) acts as an apical anchoring site for PDZK1.