The chemical inventory of the inner regions of planet-forming disks - the JWST/MINDS program.

The understanding of planet formation has changed recently, embracing the new idea of pebble accretion. This means that the influx of pebbles from the outer regions of planet-forming disks to their inner zones could determine the composition of planets and their atmospheres. The solid and molecular components delivered to the planet-forming region can be best characterized by mid-infrared spectroscopy. With Spitzer low-resolution (R = 100, 600) spectroscopy, this approach was limited to the detection of abundant molecules, such as H2O, C2H2, HCN and CO2. This contribution will present the first results of the MINDS (MIRI mid-INfrared Disk Survey, PI:Th Henning) project. Due do the sensitivity and spectral resolution provided by the James Webb Space Telescope (JWST), we now have a unique tool to obtain the full inventory of chemistry in the inner disks of solar-type stars and brown dwarfs, including also less-abundant hydrocarbons and isotopologues. The Integral Field Unit (IFU) capabilities will enable at the same time spatial studies of the continuum and line emission in extended sources such as debris disks, the flying saucer and also the search for mid-IR signatures of forming planets in systems such as PDS 70. These JWST observations are complementary to ALMA and NOEMA observations of outer-disk chemistry; together these datasets will provide an integral view of the processes occurring during the planet-formation phase.

Increased risk of infections in pediatric Fontan patients after heart transplantation.

BACKGROUND: Infectious complications are a major cause of morbidity and mortality after HT. Fontan patients may be more susceptible to post-HT infections. METHODS: This was a single-center, retrospective cohort analysis of pediatric patients undergoing HT for FF physiology or DCM, who underwent induction with ATG. The primary endpoint was an infection in the first 180 days post-HT, defined as positive (1) blood/urine/respiratory culture; (2) viral PCR; (3) skin or wound infection; and/or (4) culture-negative infection if ≥5 days of antibiotics were completed. Secondary endpoints included (1) cell counts after ATG; (2) PTLD; and (3) rejection (≥Grade 2R ACR or pAMR2) in the first 180 days post-HT. RESULTS: A total of 59 patients (26 FF, 33 DCM) underwent HT at 14.7 (IQR 10.6, 19.5) and 11.7 (IQR 1.4, 13.6) years of age, respectively. The median total ATG received was 7.4 (IQR 4.9, 7.7) vs 7.5 (IQR 7.3, 7.6) mg/kg (p = NS) for FF and DCM patients, respectively. Twenty-three patients (39%) developed an infection 180 days post-HT, with a higher rate of infection in FF patients (54% vs 27%, p = .03). Adjusted for pre-transplant absolute lymphocyte count, FF patients had a higher risk of infection at 30 days post-HT (OR 7.62, 95% CI 1.13-51.48, p = .04). There was no difference in the incidence of PTLD (12% vs 0%; p = .08) or rejection (12% vs 21%; p = .49). CONCLUSION: Compared to DCM patients, FF patients have a higher risk of infection. Modifications to induction therapy for FF patients should be considered.

Re-evaluating the need for chronic toxicity studies with therapeutic monoclonal antibodies, using a weight of evidence approach.

To support registration of monoclonal antibodies (mAbs) for chronic indications, 6-month toxicity studies have historically been conducted. Experience with mAb development has shown a relatively benign and well-understood safety profile for this class, with most toxicity findings anticipated based on pharmacology. We evaluated whether a 6-month toxicity study is necessary to assess the long-term safety of mAbs. Data on First-in-Human (FIH)-enabling and chronic toxicity studies were shared for 142 mAbs submitted by 11 companies. Opportunities to further optimize study designs to reduce animal usage were identified. For 71% of mAbs, no toxicities or no new toxicities were noted in chronic studies compared to FIH-enabling study findings. New toxicities of potential concern for human safety or that changed trial design were identified in 13.5% of cases, with 7% being considered critical and 2% leading to program termination. An iterative, weight-of-evidence model which considers factors that influence the overall risk for a mAb to cause toxicity was developed. This model enables an evidence-based justification, suggesting when 3-month toxicity studies are likely sufficient to support late-stage clinical development and registration for some mAbs.

The use of recovery animals in nonclinical safety assessment studies with monoclonal antibodies: further 3Rs opportunities remain.

Assessment of reversibility from nonclinical toxicity findings in animals with potential adverse clinical impact is required during pharmaceutical development, but there is flexibility around how and when this is performed and if recovery animals are necessary. For monoclonal antibodies (mAbs) and in accordance with ICH S6(R1) if inclusion of recovery animals is warranted, this need only occur in one study. Data on study designs for first-in-human (FIH)-enabling and later-development toxicity studies were shared from a recent collaboration between the NC3Rs, EPAA, Netherlands Medicines Evaluation Board (MEB) and 14 pharmaceutical companies. This enabled a review of practices on recovery animal use during mAb development and identification of opportunities to reduce research animal use. Recovery animals were included in 68% of FIH-enabling and 69% of later-development studies, often in multiple studies in the same program. Recovery groups were commonly in control plus one test article-dosed group or in all dose groups (45% of studies, each design). Based on the shared data review and conclusions, limiting inclusion of recovery to a single nonclinical toxicology study and species, study design optimisation and use of existing knowledge instead of additional recovery groups provide opportunities to further reduce animal use within mAb development programs.

Navigating Trauma Patients and Families Through Unfamiliar Territory.

BACKGROUND: Trauma teams work diligently to manage the complex medical needs of trauma patients. In addition to medical care, there is also a need to assist patients and their families as they navigate the emotional and physical journey of trauma. The role of trauma nurse navigator was developed to address these holistic needs. OBJECTIVE: This article aims to describe the implementation of a trauma nurse navigator role. METHODS: This article describes the development and implementation of a trauma nurse navigator role at a Level II trauma center in 2018. The trauma nurse navigator serves as a patient resource and utilizes creative problem solving to optimize care. The trauma nurse navigator also serves on the multidisciplinary team, working with providers, nursing staff, rehabilitation staff, and case management to provide seamless care to trauma patients. RESULTS: Implementation of the trauma nurse navigator role was well received by patients, families, and the multidisciplinary trauma team. The trauma nurse navigator role expanded the psychosocial support of trauma patients and increased patient satisfaction. CONCLUSION: Although other specialties have seen the benefits of including a patient navigator on the team, this is a potential for trauma centers as they strive to provide high-quality patient care.

Postintensive Care Syndrome: Feasibly Bridging Care at a Tertiary Trauma Center.

BACKGROUND: Advancements in critical care management have improved mortality rates of trauma patients; however, research has identified physical and psychological impairments that remain with patients for an extended time. Cognitive impairments, anxiety, stress, depression, and weakness in the postintensive care phase are an impetus for trauma centers to examine their ability to improve patient outcomes. OBJECTIVE: This article describes one center's efforts to intervene to address postintensive care syndrome in trauma patients. METHODS: This article describes implementing aspects of the Society of Critical Care Medicine's liberation bundle to address postintensive care syndrome in trauma patients. RESULTS: The implementation of the liberation bundle initiatives was successful and well received by trauma staff, patients, and families. It requires strong multidisciplinary commitment and adequate staffing. Continued focus and retraining are requirements in the face of staff turnover and shortages, which are real-world barriers. CONCLUSIONS: Implementation of the liberation bundle was feasible. Although the initiatives were positively received by trauma patients and their families, we identified a gap in the availability of long-term outpatient services for trauma patients after discharge from the hospital.

Multiplexed Quantitative Analysis of Antibody-Drug Conjugates with Labile CBI-Dimer Payloads In Vivo Using Immunoaffinity LC-MS/MS.

Quantitative analysis of antibody-drug conjugates (ADCs) involves cleavage of ADCs into smaller analytes representing different components and subsequent measurements from multiple assays for a more comprehensive pharmacokinetic (PK) assessment. Multiple PK analytes including the drug remaining conjugated to the antibody (or antibody-conjugated drug, acDrug) and total antibody can be accessed simultaneously using a multiplex assay by proteolytic digestion of an ADC, if the sites of conjugation are homogeneous for an ADC and the linker drug is stable to proteases. Herein, a multiplexed immunoaffinity liquid chromatography-mass spectrometry (LC-MS)/MS PK assay is described involving immunoaffinity enrichment, enzymatic conversion of prodrug, trypsin digestion, and LC-MS/MS as applied to next-generation ADCs constructed from linker drugs bearing dimeric cyclopropabenzindole (CBI) payloads (duocarmycin analogues). The cytotoxic payload is chemically labile, requiring extensive optimization in sample preparation steps to stabilize the drug without ex vivo modification and to convert the prodrug into a single active form of the drug. The qualification data for this assay format showed that this approach provides robust acDrug and total antibody data and can be extended to ADCs with different monoclonal antibody frameworks and linker chemistries. Applications of this multiplexed assay to support preclinical studies are presented.

Toxicologic Pathology Forum: Opinion on Not Euthanizing Control Animals in the Recovery Phase of Non-Rodent Toxicology Studies.

Nonclinical toxicology studies that are required to support human clinical trials of new drug candidates are generally conducted in a rodent and a non-rodent species. These studies typically contain a vehicle control group and low, intermediate, and high dose test article groups. In addition, a dosing-free recovery phase is sometimes included to determine reversibility of potential toxicities observed during the dosing phase and may include additional animals in the vehicle control and one or more dose groups. Typically, reversibility is determined by comparing the test article-related changes in the dosing phase animals to concurrent recovery phase animals at the same dose level. Therefore, for interpretation of reversibility, it is not always essential to euthanize the recovery vehicle control animals. In the absence of recovery vehicle control tissues, the pathologist's experience, historical control database, digital or glass slide repositories, or literature can be used to interpret the findings in the context of background pathology of the species/strain/age. Therefore, in most studies, the default approach could be not to euthanize recovery vehicle control animals. This article provides opinions on scenarios that may or may not necessitate euthanasia of recovery phase vehicle control animals in nonclinical toxicology studies involving dogs and nonhuman primates.

Fatal nocardiosis infection in a pediatric patient with an immunodeficiency after heart re-transplantation.

BACKGROUND: Nocardia infections are rare opportunistic infections in SOT recipients, with few reported pediatric cases. Pediatric patients with single ventricle congenital heart defects requiring HT may be more susceptible to opportunistic infections due to a decreased T-cell repertoire from early thymectomy and potential immunodeficiencies related to their congenital heart disease. Other risk factors in SOT recipients include the use of immunosuppressive medications and the development of persistent lymphopenia, delayed count recovery and/or lymphocyte dysfunction. METHODS: We report the case of a patient with hypoplastic left heart syndrome who underwent neonatal congenital heart surgery (with thymectomy) prior to palliative surgery and 2 HTs. RESULTS: After developing respiratory and neurological symptoms, the patient was found to be positive for Nocardia farcinica by BAL culture and cerebrospinal fluid PCR. Immune cell phenotyping demonstrated an attenuated T and B-cell repertoire. Despite antibiotic and immunoglobulin therapy, his symptoms worsened and he was subsequently discharged with hospice care. CONCLUSION: Pediatric patients with a history of congenital heart defects who undergo neonatal thymectomy prior to heart transplantation and a long-term history of immunosuppression should undergo routine immune system profiling to evaluate for T- and B-cell deficiency as risk factors for opportunistic infection. Such patients could benefit from long-term therapy with TMP/SMX for optimal antimicrobial prophylaxis, with desensitization as needed for allergies. Disseminated nocardiosis should be considered when evaluating acutely ill SOT recipients, especially those with persistent lymphopenia and known or suspected secondary immunodeficiencies.

Increasing the Reuse of Protein Non-Naïve Nonhuman Primates in Pharmaceutical Drug Discovery and Development: An Overview and Industry Position on the Challenges and Benefits.

The IQ Consortium NHP Reuse Working Group (WG) comprises members from 15 pharmaceutical and biotechnology companies. In 2020, the WG developed and distributed a detailed questionnaire on protein non-naïve NHP reuse to the WG member companies. The WG received responses from key stakeholders including principal investigators, facility managers, animal welfare officers and research scientists. This paper's content reflects the consolidated opinion of the WG members and the questionnaire responses on the subject of NHP reuse within nonclinical programs at all stages of research and development. Many of the pharmaceutical companies represented in the working group or participating in the questionnaire have already achieved some level of NHP reuse in their nonclinical programs, but the survey results suggested that there is significant potential to increase NHP reuse further and a need to understand the considerations involved in reuse more clearly. The WG has also focused carefully on the inherent concerns and risks of implementing protein non-naive NHP reuse and has evaluated the best methods of risk assessment and decision-making. This paper presents a discussion on the challenges and opportunities surrounding protein non-naïve NHP reuse and aims to stimulate further industry dialogue on the subject and provide guidance for pharmaceutical companies to establish roadmaps and decision trees enabling increased protein non-naïve NHP reuse. In addition, this paper represents a solid basis for collaborative engagement between pharmaceutical and biotechnology companies with contract research organizations (CROs) to discuss how the availability of protein non-naïve NHP within CROs can be better leveraged for their use within nonclinical studies.

Antibody-Drug Conjugates Derived from Cytotoxic seco-CBI-Dimer Payloads Are Highly Efficacious in Xenograft Models and Form Protein Adducts In Vivo.

This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.

An IQ Consortium Perspective on The Scientific Committee on Health, Environmental and Emerging Risks Final Opinion on the Need for Nonhuman Primates in Biomedical Research, Production and Testing of Products and Devices (Update 2017).

The recent Scientific Committee on Health, Environmental and Emerging Risks Final Opinion on "The need for nonhuman primates in biomedical research, production and testing of products and devices" (2017 SCHEER) highlights approaches that could significantly contribute to the replacement, reduction, and refinement of nonhuman primate (NHP) studies. Initiatives that have the potential to affect NHP welfare and/or their use are expected to be appropriate, fair, and objective and publicly disseminated information focused on NHPs in biomedical research, which includes toxicologic and pathologic research and testing, should be objectively evaluated by stakeholder scientists, researchers, and veterinarians. Thus, IQ Consortium member companies convened to develop an informed and objective response, focusing on identifying areas of agreement, potential gaps, or missing information in 2017 SCHEER. Overall, the authors agree that many positions in the 2017 SCHEER Opinion generally align with industry views on the use of NHPs in research and testing, including the ongoing need of NHPs in many areas of research. From the perspective of the IQ Consortium, there are several topics in the 2017 SCHEER that merit additional comment, attention, or research, as well as consideration in future opinions.

Healthy hearts in pediatric heart transplant patients with an exercise and diet intervention via live video conferencing-Design and rationale.

BACKGROUND: Pediatric heart transplant (PedHtx) patients have increased cardiovascular risk profiles that affect their long-term outcomes and quality of life. We designed a 12- to 16-week diet and exercise intervention delivered via live video conferencing to improve cardiovascular health. Our methodology and baseline assessment of the first 13 enrolled patients are reported. METHODS: Inclusion criteria are as follows: (a) 8-19 years old; (b) heart transplant >12 months; (c) ability to fast overnight; (d) cardiac clearance by cardiologist; and (e) presence of an adult at home during exercise sessions for patients <14 years old. Exclusion criteria are as follows: (a) acute illness; (b) latex allergy; (c) transplant rejection <3 months ago; and (d) multi-organ transplantation. The intervention consists of one diet and three exercise sessions weekly via live video conferencing. Study visits are conducted at baseline, intervention completion, and end of maintenance period. RESULTS: A total of 13 participants (15.2 [2.3] years) have been enrolled. Median percent-predicted VO2 max was 56.8 [20.7]% (10 patients <70%). Ten patients had abnormal endothelial function (reactive hyperemia index <1.9; 1.4 [0.325]) and 11 patients had stiff arteries (pulse wave velocity ≧5.5 m/s for 15-19 years, ≧4.5 m/s for 8-14 years; 5.6 [0.7] m/s). Patients had suboptimal diets (saturated fat: 22.7 [23.8] g/d, sodium: 2771 [1557] mg/d) and were sedentary at a median of 67.5 [13.8]% of their time. CONCLUSIONS: Baseline assessment confirms that PedHtx patients have abnormal cardiac, vascular, and functional health indices, poor dietary habits, and are sedentary. These results support the rationale to test the feasibility and impact of a non-pharmacologic lifestyle intervention in this patient population.

A Tiered Approach to Trauma Education in the Emergency Department.

Although trauma centers are required to provide trauma education to nurses caring for trauma patients, there are no clearly defined standards for this education. In an effort to improve emergency department (ED) trauma nursing care, a tiered approach to ED trauma education (basic, intermediate, and advanced) was developed to provide specialized trauma education to a larger number of ED nurses at a Level II trauma center in Georgia. This tiered approach to ED trauma nurse education has resulted in the ability to quickly activate multiple trauma teams that work together competently and efficiently, leading to improved patient care and development of competent ED trauma nurses.

Platelet proteins cause basophil histamine release through an immunoglobulin-dependent mechanism.

BACKGROUND: A general understanding of allergic transfusion reaction mechanisms remains elusive. Multiple mechanisms have been proposed, but none have been compared experimentally. STUDY DESIGN AND METHODS: We used histamine release (HR) from healthy human donor basophils to model allergic transfusion reactions. Platelet component supernatant (plasma), platelet lysate, and manipulated platelet lysates (dialyzed, delipidated, trypsinized, mild heat-inactivated, and ultracentrifuged) were used to characterize allergic stimuli. Immunoglobulin-dependent mechanisms were investigated through cell surface immunoglobulin depletion and ibrutinib signaling inhibition. HR induced by platelet mitochondria was compared with HR by platelet lysate with or without DNase treatment. RESULTS: Robust, dose-responsive HR to platelet lysate was observed in two of eight nulliparous, never-transfused, healthy donors. No HR was observed with plasma. Among manipulated platelet lysates, only trypsin treatment significantly reduced HR (39% reduction; p = 0.008). HR in response to platelet lysate significantly decreased with either cell surface immunoglobulin depletion or ibrutinib pretreatment. Platelet mitochondria induced minimal basophil HR, and DNase treatment did not inhibit platelet lysate-induced HR. CONCLUSION: Type I immediate hypersensitivity to platelet proteins may be an allergic transfusion reaction mechanism. Prior sensitization to human proteins is not required for basophil responses to platelet proteins. Further study into the relative contributions of hypersensitivity to platelet versus plasma proteins in transfusion is warranted.

Group visits in the pediatric heart transplant outpatient clinic.

The "GVM" has emerged as an alternative to traditional individualized appointments in the ambulatory care setting. We hypothesized that group visits could successfully be utilized in a PHtx clinic. Seven patients, ages 1-18 yr old, and their families participated in a total of 11 group visits in lieu of individualized appointments. Patients were divided into two groups based on whether they were greater or less than one yr post-transplant. Patient/provider satisfaction, medication adherence, and content retention were ascertained via questionnaires and free-response tests. Total clinic throughput time, including per-patient clinic utilization time, was compared to historical data. Six of seven patients completed the study with one dropout. Overall satisfaction ratings were 3.98 of 4 with all patients reporting that they would "strongly recommend" group visits to others. Health information retention tests demonstrated improvement between pre- and post-tests in eight of nine (89%) of the group visits. Overall clinic utilization decreased by nearly 50% while providing 70 min of face-to-face time with the provider. Medication adherence neared 100% for all patients. The GVM can be successfully applied to the PHtx population with high patient and provider satisfaction, more face-to-face time, excellent content retention, and greatly improved clinic efficiency.

Increasing plasma [K+] by intravenous potassium infusion reduces NCC phosphorylation and drives kaliuresis and natriuresis.

Dietary potassium loading results in rapid kaliuresis, natriuresis, and diuresis associated with reduced phosphorylation (p) of the distal tubule Na(+)-Cl(-) cotransporter (NCC). Decreased NCC-p inhibits NCC-mediated Na(+) reabsorption and shifts Na(+) downstream for reabsorption by epithelial Na(+) channels (ENaC), which can drive K(+) secretion. Whether the signal is initiated by ingesting potassium or a rise in plasma K(+) concentration ([K(+)]) is not understood. We tested the hypothesis, in male rats, that an increase in plasma [K(+)] is sufficient to reduce NCC-p and drive kaliuresis. After an overnight fast, a single 3-h 2% potassium (2%K) containing meal increased plasma [K(+)] from 4.0 ± 0.1 to 5.2 ± 0.2 mM; increased urinary K(+), Na(+), and volume excretion; decreased NCC-p by 60%; and marginally reduced cortical Na(+)-K(+)-2Cl(-) cotransporter (NKCC) phosphorylation 25% (P = 0.055). When plasma [K(+)] was increased by tail vein infusion of KCl to 5.5 ± 0.1 mM over 3 h, significant kaliuresis and natriuresis ensued, NCC-p decreased by 60%, and STE20/SPS1-related proline alanine-rich kinase (SPAK) phosphorylation was marginally reduced 35% (P = 0.052). The following were unchanged at 3 h by either the potassium-rich meal or KCl infusion: Na(+)/H(+) exchanger 3 (NHE3), NHE3-p, NKCC, ENaC subunits, and renal outer medullary K(+) channel. In summary, raising plasma [K(+)] by intravenous infusion to a level equivalent to that observed after a single potassium-rich meal triggers renal kaliuretic and natriuretic responses, independent of K(+) ingestion, likely driven by decreased NCC-p and activity sufficient to shift sodium reabsorption downstream to where Na(+) reabsorption and flow drive K(+) secretion.

Sialic Acid Mediated Endothelial and Hepatic Uptake: A Mechanism based Mathematic Model Elucidating the Complex Pharmacokinetics and Pharmacodynamics of Efmarodocokin Alfa, a Variably Glycosylated Fusion Protein.

Sialic acid (SA) is crucial for protecting glycoproteins from clearance. Efmarodocokin alfa (IL-22Fc), a fusion protein agonist that links IL-22 to the crystallizable fragment (Fc) of human IgG4, contains 8 N-glycosylation sites and exhibits heterogeneous and variable terminal sialylation biodistribution. This presents a unique challenge for Pharmacokinetic (PK) and Pharmacodynamic (PD) analysis and cross-species translation. In this study, we sought to understand how varying SA levels and heterogeneous distribution contribute to IL-22Fc's complex PKPD properties. We initially used homogenous drug material with varying SA levels to examine PKPD in mice. Population PKPD analysis based on mouse data revealed that SA was a critical covariate simultaneously accounting for the substantial between subject variability (BSV) in clearance (CL), distribution clearance (CLd), and volume of distribution (Vd). In addition to the well-established mechanism by which SA inhibits ASGPR activity, we hypothesized a novel mechanism by which decrease in SA increases the drug uptake by endothelial cells. This decrease in SA, leading to more endothelial uptake, was supported by the neonatal Fc receptor (FcRn) dependent cell-based transcytosis assay. The population analysis also suggested in vivo EC50 (IL-22Fc stimulating Reg3ß) was independent on SA, while the in-vitro assay indicated a contradictory finding of SA-in vitro potency relationship. We created a mechanism based mathematical (MBM) PKPD model incorporating the decrease in SA mediated endothelial and hepatic uptake, and successfully characterized the SA influence on IL-22Fc PK, as well as the increased PK exposure being responsible for increased PD. Thereby, the MBM model supported that SA has no direct impact on EC50, aligning with the population PKPD analysis. Subsequently, using the MBM PKPD model, we employed 5 subpopulation simulations to reconstitute the heterogeneity of drug material. The simulation accurately predicted the PKPD of heterogeneously and variably sialylated drug in mouse, monkey and human. The successful prospective validation confirmed the MBM's ability to predict IL-22Fc PK across variable SA levels, homogenous to heterogeneous material, and across species (R2=0.964 for clearance prediction). Our model prediction suggests an average of 1 mol/mol SA increase leads to a 50% increase in drug exposure. This underlines the significance of controlling sialic acid levels during lot-to-lot manufacturing.

Effects of ACE inhibition and ANG II stimulation on renal Na-Cl cotransporter distribution, phosphorylation, and membrane complex properties.

The renal distal tubule Na-Cl cotransporter (NCC) reabsorbs <10% of the filtered Na(+) but is a key control point for blood pressure regulation by angiotensin II (ANG II), angiotensin-converting enzyme inhibitors (ACEI), and thiazide diuretics. This study aimed to determine whether NCC phosphorylation (NCCp) was regulated by acute (20-30 min) treatment with the ACEI captopril (12 µg/min × 20 min) or by a sub-pressor dose of ANG II (20 ng·kg(-1)·min(-1)) in Inactin-anesthetized rats. By immuno-EM, NCCp was detected exclusively in or adjacent to apical plama membranes (APM) in controls and after ACEI or ANG II treatment, while NCC total was detected in both APM and subapical cytoplasmic vesicles (SCV) in all conditions. In renal homogenates, neither ACEI nor ANG II treatment altered NCCp abundance, assayed by immunoblot. However, by density gradient fractionation we identified a pool of low-density APM in which NCCp decreased 50% in response to captopril and was restored during ANG II infusion, and another pool of higher-density APM that responded reciprocally, indicative of regulated redistribution between two APM pools. In both pools, NCCp was preferentially localized to Triton-soluble membranes. Blue Native gel electrophoresis established that APM NCCp localized to ~700 kDa complexes (containing γ-adducin) while unphosphorylated NCC in intracellular membranes primarily localized to ~400 kDa complexes: there was no evidence for native monomeric or dimeric NCC or NCCp. In summary, this study demonstrates that phosphorylated NCC, localized to multimeric complexes in the APM, redistributes in a regulated manner within the APM in response to ACEI and ANG II.

Differential regulation of Na+ transporters along nephron during ANG II-dependent hypertension: distal stimulation counteracted by proximal inhibition.

During angiotensin II (ANG II)-dependent hypertension, ANG II stimulates, while hypertension inhibits, Na(+) transporter activity to balance Na(+) output to input. This study tests the hypothesis that ANG II infusion activates Na(+) transporters in the distal nephron while inhibiting transporters along the proximal nephron. Male Sprague-Dawley rats were infused with ANG II (400 ng·kg(-1)·min(-1)) or vehicle for 2 wk. Kidneys were dissected (cortex vs. medulla) or fixed for immunohistochemistry (IHC). ANG II increased mean arterial pressure by 40 mmHg, urine Na(+) by 1.67-fold, and urine volume by 3-fold, evidence for hypertension and pressure natriuresis. Na(+) transporters' abundance and activation [assessed by phosphorylation (-P) or proteolytic cleavage] were measured by immunoblot. During ANG II infusion Na(+)/H(+) exchanger 3 (NHE3) abundance decreased in both cortex and medulla; Na-K-2Cl cotransporter 2 (NKCC2) decreased in medullary thick ascending loop of Henle (TALH) and increased, along with NKCC2-P, in cortical TALH; Na-Cl cotransporter (NCC) and NCC-P increased in the distal convoluted tubule; and epithelial Na(+) channel subunits and their cleaved forms were increased in both cortex and medulla. Like NKCC2, STE20/SPS1-related proline alanine-rich kinase (SPAK) and SPAK-P were decreased in medulla and increased in cortex. By IHC, during ANG II NHE3 remained localized to proximal tubule microvilli at lower abundance, and the differential regulation of NKCC2 and NKCC2-P in cortex versus medulla was evident. In summary, ANG II infusion increases Na(+) transporter abundance and activation from cortical TALH to medullary collecting duct while the hypertension drives a natriuresis response evident as decreased Na(+) transporter abundance and activation from proximal tubule through medullary TALH.