Structure-function relationships in the sodium chloride cotransporter.

The thiazide sensitive Na+:Cl- cotransporter (NCC) is the principal via for salt reabsorption in the apical membrane of the distal convoluted tubule (DCT) in mammals and plays a fundamental role in managing blood pressure. The cotransporter is targeted by thiazide diuretics, a highly prescribed medication that is effective in treating arterial hypertension and edema. NCC was the first member of the electroneutral cation-coupled chloride cotransporter family to be identified at a molecular level. It was cloned from the urinary bladder of the Pseudopleuronectes americanus (winter flounder) 30 years ago. The structural topology, kinetic and pharmacology properties of NCC have been extensively studied, determining that the transmembrane domain (TM) coordinates ion and thiazide binding. Functional and mutational studies have discovered residues involved in the phosphorylation and glycosylation of NCC, particularly on the N-terminal domain, as well as the extracellular loop connected to TM7-8 (EL7-8). In the last decade, single-particle cryogenic electron microscopy (cryo-EM) has permitted the visualization of structures at high atomic resolution for six members of the SLC12 family (NCC, NKCC1, KCC1-KCC4). Cryo-EM insights of NCC confirm an inverted conformation of the TM1-5 and TM6-10 regions, a characteristic also found in the amino acid-polyamine-organocation (APC) superfamily, in which TM1 and TM6 clearly coordinate ion binding. The high-resolution structure also displays two glycosylation sites (N-406 and N-426) in EL7-8 that are essential for NCC expression and function. In this review, we briefly describe the studies related to the structure-function relationship of NCC, beginning with the first biochemical/functional studies up to the recent cryo-EM structure obtained, to acquire an overall view enriched with the structural and functional aspects of the cotransporter.

Going Further: Comprehensive Disease Control of Rheumatoid Arthritis, Targeting Cytokines and Chemokines.

OBJECTIVES: Mechanism of action of biological and synthetic disease-modifying antirheumatic drugs (DMARDs) includes the inhibition of specific proinflammatory cytokines. This study aimed to elucidate the cytokines and chemokines inhibited by different treatments (conventional synthetic DMARD [csDMARD], biological and targeted synthetic DMARD) in rheumatoid arthritis (RA). METHODS: Fifty-nine RA patients with low disease activity or remission included in a cross-sectional study were classified by treatment in groups: abatacept, certolizumab, rituximab (RTX), tocilizumab, tofacitinib (TOF), baricitinib (BAR), and csDMARD. Cytokine and chemokine serum levels were measured by LEGENDplex Human Inflammation panel. Quantitative variables were compared using Student t or Mann-Whitney U test as appropriate, whereas qualitative variables were compared using χ2 or Fisher exact test. p < 0.05 was considered significant. RESULTS: Certolizumab, RTX, tocilizumab, and TOF showed that most cytokine pathways inhibited: tumor necrosis factor α, interferon γ, interleukin 1ß (IL-1ß), IL-12, IL-18, and IL-23; in addition, csDMARDs showed a similar inhibition patron except for IL-23. Serum level of tumor necrosis factor α pathway was one of the most inhibited being undetectable in RTX, TOF, and BAR groups. Interleukin 6 was shown to be inhibited by abatacept, RTX, and TOF; however, higher levels were observed in 3 patients treated with tocilizumab. Abatacept, certolizumab, RTX, and TOF downregulated IL-10 in this group of patients but remained detectable in almost half of the subjects, with the highest levels in the BAR group. The active pathways that remained the most were CC chemokine ligand 2, IL-8, IL-17, and IL-33. CONCLUSIONS: Understanding the cytokine chemokine pathways inhibition could help rheumatologists to prescribe a tailored therapy using the arsenal of DMARDs for individualized RA treatment in an evidence-based decision manner.

WNK3 and WNK4 exhibit opposite sensitivity with respect to cell volume and intracellular chloride concentration.

Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl-]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl-]i, respectively. Cell shrinkage and a decrease in [Cl-]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl-]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl-]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl-]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl-]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK's carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.

Common errors in dual-energy X-ray absorptiometry scans in imaging centers in Ecuador.

Dual-energy X-ray absorptiometry is recognized for measuring bone mineral density. The lack of knowledge can lead to errors both in the acquisition of information and in its analysis and subsequent interpretation. The main errors in Ecuadorian Centers were positioning of the patient to the equipment and incorrect analyzed area. PURPOSE/INTRODUCTION: Dual-energy X-ray absorptiometry (DXA) is recognized as the gold standard for measuring bone mineral density (BMD) with acceptable errors, good precision, and reproducibility. However, the training of operators in different centers and countries is not standardized, and the lack of knowledge can lead to errors both in the acquisition of information and in its analysis and subsequent interpretation. The purpose was to determine the most common errors in the performance of bone densitometry from different imaging centers in Ecuador. METHODS: Cross-sectional descriptive study. We collected DXA scans from different imaging centers in Ecuador. Data from the DXA scan included city of origin, type of specialist that requested it, and densitometry diagnosis. The DXA images provided were analyzed double blind by experts in the field from Argentina. RESULTS: From a total of 141 patients with a mean age of 61 ± 10 years, 93.6% were women. About 78% of the DXA scans came from private imaging centers and 22% from public centers, 95% of all came from the city of Guayaquil. The machines used were Hologic 50.4% and Lunar 49.6%. The densitometric diagnosis was 16.3% normal, 46.1% osteoporosis, and 37.6% osteopenia. A total of 112 left hip and 49 right hip scans were analyzed from which 31.2% and 22.4% had errors in patient positioning, respectively, mainly internal or external rotation. About 140 lumbar scans were analyzed from which 21.4% had patient positioning errors (not centered or not straight). Also in 38.5% the vertebral area did not correspond to L1-L4. About 3.5% had artifacts such as a metal bar or implant. The region of interest was misplaced in 24.1% of the lumbar scans and 19.9% of the femur. CONCLUSIONS: DXA quality standards exist but are often not implemented in clinical practice. When studies are performed incorrectly, it can lead to important errors in diagnosis and therapy. Physicians interested in the management of osteoporosis, although not directly involved in the performance and interpretation of DXA, should be familiar with the protocols to minimize errors and allow the proper use of bone densitometry.

Revisiting Keratoconjunctivitis sicca associated with Human T-Cell Lymphotropic Virus Type 1: prevalence, clinical aspects and proviral load.

BACKGROUND: The prevalence of keratoconjunctivitis sicca (KCS) associated with Human T-Cell Lymphotropic Virus Type 1 (HTLV-1) (HTLV-1/KCS) has been estimated at around 37%, but its clinical manifestations are poorly described. PURPOSE: To determine the prevalence and associated factors of HTLV-1/KCS in a large cohort of HTLV-1-infected individuals living in Salvador, Brazil. METHODS: A cross-sectional study was conducted between June 2004 and September 2017 at the Integrative and Multidisciplinary Center for HTLV in Salvador, Bahia-Brazil. Data from 758 HTLV-1-infected patients was collected. A complete ophthalmologic examination was performed in both eyes. Lacrimal function was evaluated by breakup time, Rose Bengal and Schirmer I Tests. KCS diagnosis was considered in the presence of at least two out of three positive tests. HTLV-1 proviral load Crude and Adjusted Prevalence Rates (PR) with 95% Confidence Intervals (95% CI) were estimated using multivariate Poisson Regression with robust error variance. RESULTS: The overall prevalence of KCS was 31.7%, with higher rates observed in HTLV-1-associated myelopathy/tropical spastic paraparesis patients (crude PR: 1.84; CI95%: 1.50-2.26) even after adjusting for age, sex, time of HTLV-1 diagnosis and schooling (adjusted PR: 1.63; CI95%: 1.31-2.02). Proviral load, low corrected visual acuity, burning and/or pain and itching were all significantly higher in patients with KCS. CONCLUSION: Burning and/or pain and itching and low corrected visual acuity were the most common alterations of HTLV-1/KCS. High Proviral load was found to be associated with the presence of KCS. It is strongly recommended that HTLV-1 patients undergo periodic ophthalmologic examination to promote the early diagnosis of KCS and prevent the consequences associated with dry eye disease.

Revisiting Keratoconjunctivitis sicca associated with Human T-Cell Lymphotropic Virus Type 1: prevalence, clinical aspects and proviral load

ABSTRACT Background: The prevalence of keratoconjunctivitis sicca (KCS) associated with Human T-Cell Lymphotropic Virus Type 1 (HTLV-1) (HTLV-1/KCS) has been estimated at around 37%, but its clinical manifestations are poorly described. Purpose: To determine the prevalence and associated factors of HTLV-1/KCS in a large cohort of HTLV-1-infected individuals living in Salvador, Brazil. Methods: A cross-sectional study was conducted between June 2004 and September 2017 at the Integrative and Multidisciplinary Center for HTLV in Salvador, Bahia-Brazil. Data from 758 HTLV-1-infected patients was collected. A complete ophthalmologic examination was performed in both eyes. Lacrimal function was evaluated by breakup time, Rose Bengal and Schirmer I Tests. KCS diagnosis was considered in the presence of at least two out of three positive tests. HTLV-1 proviral load Crude and Adjusted Prevalence Rates (PR) with 95% Confidence Intervals (95% CI) were estimated using multivariate Poisson Regression with robust error variance. Results: The overall prevalence of KCS was 31.7%, with higher rates observed in HTLV-1-associated myelopathy/tropical spastic paraparesis patients (crude PR: 1.84; CI95%: 1.50-2.26) even after adjusting for age, sex, time of HTLV-1 diagnosis and schooling (adjusted PR: 1.63; CI95%: 1.31-2.02). Proviral load, low corrected visual acuity, burning and/or pain and itching were all significantly higher in patients with KCS. Conclusion: Burning and/or pain and itching and low corrected visual acuity were the most common alterations of HTLV-1/KCS. High Proviral load was found to be associated with the presence of KCS. It is strongly recommended that HTLV-1 patients undergo periodic ophthalmologic examination to promote the early diagnosis of KCS and prevent the consequences associated with dry eye disease.

Subclinical parameters of arterial stiffness and arteriosclerosis correlate with QRISK3 in systemic lupus erythematosus.

It is well known that cardiovascular diseases (CVD) are a major contributor of death in systemic lupus erythematosus (SLE) as well in other rheumatic illness. In the last decades, there has been a growing development of different methodologies with the purpose of early detection of CVD. OBJECTIVE: The aim of this study is to correlate the usefulness of subclinical parameters of vascular aging and QRISK 3-2017 score for early detection of CVD in SLE. METHODS: Clinical assessment including systemic lupus erythematosus disease activity index (SLEDAI) and systemic lupus international collaborating clinics / american college of rheumatology damage index (SLICC/ACR DI), laboratory measurements, carotid ultrasound examination, carotid intima media thickness (cIMT) measurement, carotid distention and diameter analysis, arterial stiffness measurement measured by tonometry and QRISK 3-2017 were done. All results were analyzed by SPSS 24 software. RESULTS: We observed correlation between QRISK3 and mean cIMT (rs = 0.534, P < 0.001), PWV (rs = 0.474, P < 0.001), cfPWV (rs = 0.569, P < 0.001) and distensibility (rs = -0.420, P = 0.006). Consistent with above, SLE patients in middle and high risk QRISK 3-2017 showed increased arterial stiffness versus low risk group. CONCLUSIONS: We encourage to the rheumatology community to assess cardiovascular risk in SLE patients with QRISK 3-2017 risk calculator as an alternative method at the outpatient clinic along a complete cardiovascular evaluation when appropriate.

C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity.

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overactivation of the renal Na+/Cl- cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4-/- mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4's C terminus at several positions increased its kinase activity toward Ste20-related proline/alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-binding site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2+-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.

Validez concurrente del examen clínico objetivo estructurado con el portafolio electrónico, examen teórico y promedio ponderado en estudiantes de cirugía de la Universidad Privada Antenor Orrego / Concurrent validity of the objective structured clinical examination with electronic portfolio, theoretical exam and weighted average in students of surgery at the Antenor Orrego Private University

Sujetos y métodos: Este estudio correlacional descriptivo tuvo como objetivo demostrar la validez concurrente del examen clínico objetivo estructurado (ECOE) con el promedio ponderado, la nota teórica y el portafolio electrónico en 123 estudiantes de medicina del curso de Cirugía I del IX ciclo, durante el semestre académico 2014-I, de la Facultad de Medicina de la Universidad Privada Antenor Orrego (Trujillo, Perú). Resultados: Hubo correlación bivariada aceptable (r = 0,65) entre la nota teórica y el ECOE; correlación moderada (r = 0,52) entre el promedio ponderado y el ECOE; y correlación alta (r = 0,77) entre la nota del portafolio electrónico y el ECOE. Hubo correlación lineal múltiple entre el portafolio, el ECOE y el examen teórico (coeficiente de determinación múltiple R2 = 0,55). Conclusión: Este estudio demuestra que el ECOE, el portafolio electrónico y la nota teórica utilizados para evaluar a los estudiantes de un curso de cirugía de pregrado tuvieron validez concurrente en el rango de aceptable a alta, y esta información constituye la base para mejorar los estándares de evaluación

Subjects and methods: This descriptive correlational study aimed to demonstrate the concurrent validity of the objective structured clinical examination (OSCE) with the weighted average, theoretical note and the electronic portfolio of 123 medical students of the I Course of Surgery of the ninth cycle during the academic semester 2014-I, at the Faculty of Medicine of the Antenor Orrego Private University (Trujillo, Peru). Results: There was an acceptable bivariate correlation (r = 0.65) between the theoretical note and OSCE; moderate correlation (r = 0.52) between the weighted average and OSCE; and high correlation (r = 0.77) between the notes of the electronic portfolio and OSCE. There was a multiple linear correlation among the portfolio, OSCE and theoretical note (multiple determination coefficient R2 = 0.55). Conclusion: This study showed that the OSCE, electronic portfolio and theoretical note used to evaluate students in an undergraduate course of surgery had concurrent validity in the range of acceptable and high and this information forms the basis in order to improve the standards of evaluation

The European Eel NCCß Gene Encodes a Thiazide-resistant Na-Cl Cotransporter.

The thiazide-sensitive Na-Cl cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule. NCC plays a key role in the regulation of blood pressure. Its inhibition with thiazides constitutes the primary baseline therapy for arterial hypertension. However, the thiazide-binding site in NCC is unknown. Mammals have only one gene encoding for NCC. The eel, however, contains a duplicate gene. NCCα is an ortholog of mammalian NCC and is expressed in the kidney. NCCß is present in the apical membrane of the rectum. Here we cloned and functionally characterized NCCß from the European eel. The cRNA encodes a 1043-amino acid membrane protein that, when expressed in Xenopus oocytes, functions as an Na-Cl cotransporter with two major characteristics, making it different from other known NCCs. First, eel NCCß is resistant to thiazides. Single-point mutagenesis supports that the absence of thiazide inhibition is, at least in part, due to the substitution of a conserved serine for a cysteine at position 379. Second, NCCß is not activated by low-chloride hypotonic stress, although the unique Ste20-related proline alanine-rich kinase (SPAK) binding site in the amino-terminal domain is conserved. Thus, NCCß exhibits significant functional differences from NCCs that could be helpful in defining several aspects of the structure-function relationship of this important cotransporter.