Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods

Molecular and cellular pathophysiological events occurring in the majority of rare kidney diseases remain to be elucidated. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive disorder caused by mutations in either CLDN16 or CLDN19 genes. This disease is characterized by massive urinary wasting of magnesium and calcium, osmosis deregulation and polyuria. Patients with p.G20D homozygous mutation in CLDN19 gene exhibit different progression to kidney failure suggesting that beyond the pathogenic mutation itself, other molecular events are favoring disease progression. Due to the fact that biopsy is not clinically indicated in these patients, urinary exosome-like vesicles (uEVs) can be envisioned as a valuable non-invasive source of information of events occurring in the kidney. Exosome research has increased notably to identify novel disease biomarkers but there is no consensus standardized protocols for uEVs isolation in patients with polyuria. For this reason, this work was aimed to evaluate and refine different uEVs isolation methods based on differential centrifugation, the gold standard method. Characterization by NTA, cryo-TEM and immunoblotting techniques identified the most appropriate protocol to obtain the highest yield and purest uEVs enriched fraction possible from urine control samples and FHHNC patients. Moreover, we tested five different RNA extraction methods and evaluated the miRNA expression pattern by qRT-PCR. In summary, we have standardized the conditions to proceed with the identification of differentially expressed miRNAs in uEVs of FHHNC patients, or other renal diseases characterized by polyuria

STAT3 phosphorylation at serine 727 activates specific genetic programs and promotes clear cell renal cell carcinoma (ccRCC) aggressiveness

Cancer models; Molecular medicineModelos de cáncer; Medicina molecularModels de càncer; Medicina molecularThe signal transducer and activator of transcription 3 (STAT3) is a transcription factor mainly activated by phosphorylation in either tyrosine 705 (Y705) or serine 727 (S727) residues that regulates essential processes such as cell differentiation, apoptosis inhibition, or cell survival. Aberrant activation of STAT3 has been related to development of nearly 50% of human cancers including clear cell renal cell carcinoma (ccRCC). In fact, phosho-S727 (pS727) levels correlate with overall survival of ccRCC patients. With the aim to elucidate the contribution of STAT3 phosphorylation in ccRCC development and progression, we have generated human-derived ccRCC cell lines carrying STAT3 Y705 and S727 phosphomutants. Our data show that the phosphomimetic substitution Ser727Asp facilitates a pro-tumoral phenotype in vitro, in a Y705-phosphorylation-independent manner. Moreover, we describe that STAT3 phosphorylation state determines the expression of different subsets of target genes associated with distinct biological processes, being pS727-dependent genes the most related to cellular hallmarks of cancer. In summary, the present study constitutes the first analysis on the role of overall STAT3 phosphorylation state in ccRCC and demonstrates that pS727 promotes the expression of a specific subset of target genes that might be clinically relevant as novel biomarkers and potential therapeutic targets for ccRCC.This work was supported by Ministerio de Ciencia e Innovación (Grant No. SAF201459945-R and SAF2017-89989-R) to A.M.; American Association for Cancer Research (AACR, Ref #419589) to A.M. and Red de Investigación Renal REDinREN (Grant No. 12/0021/0013) to. A.M. The group holds the Quality Mention from the Generalitat de Catalunya (Grant No. 2021 SGR 01,600). J.A. was a recipient of the Ph.D. Fellow Program from Consejo Nacional de Ciencia y Tecnología (CONACyT), México (Grant No. 549678)

Novel compound heterozygous mutations of CLDN16 in a patient with familial hypomagnesemia with hypercalciuria and nephrocalcinosis

Background: Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an autosomal recessive tubulopathy characterized by excessive urinary wasting of magnesium and calcium, bilateral nephrocalcinosis, and progressive chronic renal failure in childhood or adolescence. FHHNC is caused by mutations in CLDN16 and CLDN19, which encode the tight-junction proteins claudin-16 and claudin-19, respectively. Most of these mutations are missense mutations and large deletions are rare. Methods: We examined the clinical and biochemical features of a Spanish boy with early onset of FHHNC symptoms. Exons and flanking intronic segments of CLDN16 and CLDN19 were analyzed by direct sequencing. We developed a new assay based on Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) to investigate large CLDN16 deletions. Results: Genetic analysis revealed two novel compound heterozygous mutations of CLDN16, comprising a missense mutation, c.277G>A; p.(Ala93Thr), in one allele, and a gross deletion that lacked exons 4 and 5,c.(840+25_?)del, in the other allele. The patient inherited these variants from his mother and father, respectively. Conclusions: Using direct sequencing and our QMPSF assay, we identified the genetic cause of FHHNC in our patient. This QMPSF assay should facilitate the genetic diagnosis of FHHNC. Our study provided additional data on the genotypic spectrum of the CLDN16 gene.This work was supported by Grant PI17/00153 co-financed by the Instituto de Salud Carlos III (Spain) and the European Regional Development Fund "Another way to build Europe". Editoria

Estudi dels mecanismes genètics i moleculars associats a les diferències fenotípiques observades en pacients amb hipomagnesèmia familiar amb hipercalciúria i nefrocalcinosi (HFHNC)

La hipomagnesèmia familiar amb hipercalciúria i nefrocalcinosi (HFHNC) és una tubulopatia minoritària autosòmica recessiva causada per mutacions als gens CLDN16 o CLDN19, que codifiquen per la claudina-16 i -19, respectivament, que s'expressen a la porció gruixuda de la nansa ascendent de Henle, i estan implicades en el transport iònic paracel·lular. Aquesta malaltia es caracteritza per la pèrdua urinària massiva de calci i magnesi, nefrocalcinosi bilateral i progressió inexorable de la malaltia renal crònica, que desencadena en fallida renal. Addicionalment, la majoria de pacients amb mutacions a CLDN19 també desenvolupen alteracions oculars, ja que aquest s'expressa a les cèl·lules epitelials de la retina. Característicament, existeix una gran variabilitat fenotípica entre els pacients, inclús entre aquells que comparteixen la mutació c.59G>A; p.G20D a CLDN19 (mutació Hispànica), i entre membres d'una mateixa família. Aquest fenomen suggereix que possiblement existeixen altres processos moleculars que determinen la progressió clínica dels pacients. Sota aquesta hipòtesi, aquest treball s'ha focalitzat en l'estudi dels factors genètics i epigenètics que podrien modular la progressió de la malaltia renal. Per això, s'han obtingut les dades clíniques i mostres d'orina i sang de 30 pacients afectes d'HFHNC i 6 individus control d'arreu d'Espanya, essent la majoria dels pacients diagnosticats d'HFHNC en aquest territori. L'anàlisi de les dades clíniques va permetre avaluar les diferències en l'evolució de la malaltia renal en funció del sexe i el genotip, classificar els pacients segons la pèrdua anual de funció renal (progressió renal ràpida, moderada i lenta), identificar biomarcadors clínics de pronòstic, i evidenciar l'alta variabilitat fenotípica intrafamiliar. L'estudi de variants gèniques en homozigosi associades als fenotips més extrems (progressió ràpida i lenta) s'ha realitzat amb les dades obtingudes de la seqüenciació massiva de l'exoma dels 30 pacients de la cohort. Seguint aquesta estratègia, s'han identificat un total de 45 variants gèniques. D'entre aquestes, per la funció fisiològica dels gens on es localitzen, en destaquen la rs11207827 (al gen PATJ) i la rs1050171 (al gen EGFR). Per determinar els factors epigenètics implicats en la fisiopatologia de la HFHNC i en la seva progressió, s'han utilitzat els urinary exosome-like vesicles (uEVs) com a font no invasiva d'informació dels processos cel·lulars renals. Mitjançant microarrays, s'ha analitzat el perfil d'expressió dels miRNAs continguts als uEVs dels 20 pacients que mantenien els ronyons natius funcionals identificant-se 24 miRNAs diferencialment expressats en el conjunt de pacients respecte el grup control, i 43 en el subconjunt dels pacients homozigots per la mutació p.G20D a CLDN19. La comparació de pacients d'ambdós sexes, va mostrar únicament la infraexpressió del miR 1915 5p en aquells de sexe masculí. Per últim, s'han identificat 4 miRNAs diferencialment expressats en els pacients d'HFHNC amb progressió renal moderada, en comparació amb el de progressió lenta, i 8 en el subgrup de pacients homozigots per la mutació p.G20D a CLDN19. La biologia de sistemes i l'ús de xarxes neuronals d'intel·ligència artificial han permès associar els resultats obtinguts amb processos biològics crucials en la fisiopatologia de la HFHNC, com la fibrosi renal i el transport de calci i magnesi, entre d'altres. Aquest treball ha permès incrementar el coneixement de la fisiopatologia de la HFHNC i determinar factors clínics, genètics i epigenètics implicats en la progressió de la malaltia renal i identificar nous possibles biomarcadors pronòstics de la HFHNC, i dianes terapèutiques que podrien permetre modular la severitat de la malaltia i, en el millor dels casos, curar-la.La hipomagnesemia familiar con hipercalciuria y nefrocalcinosis (HFHNC) es una tubulopatía minoritaria autosómica recesiva causada por mutaciones en los genes CLDN16 o CLDN19, que codifican para la claudina-16 y -19, respectivamente, expresadas en la porción gruesa del asa ascendente de Henle, e implicadas en el transporte iónico paracelular. Esta enfermedad se caracteriza por la pérdida urinaria de calcio y magnesio, nefrocalcinosis bilateral y progresión inexorable de la enfermedad renal crónica, que desencadena en fallo renal. Adicionalmente, la mayoría de pacientes con mutaciones en CLDN19 también desarrollan alteraciones oculares, ya que este se expresa en las células epiteliales de la retina. Existe una gran variabilidad fenotípica entre pacientes, incluso entre aquellos que comparten la mutación c.59G>A; p.G20D en CLDN19 (mutación Hispánica), y entre miembros de una misma familia. Este fenómeno sugiere que, más allá de la mutación causante de la enfermedad, posiblemente existen otros procesos moleculares que determinan la progresión clínica de los pacientes. Bajo esta hipótesis, este trabajo se ha focalizado en el estudio de los factores genéticos y epigenéticos que podrían modular la progresión de la enfermedad renal. Para ello, se han obtenido los datos clínicos, orina y sangre de 30 pacientes afectos de HFHNC y 6 individuos control de diferentes lugares de España, siendo estos la mayoría de pacientes diagnosticados de HFHNC en este territorio. El análisis de los datos clínicos permitió evaluar las diferencias en la evolución de la enfermedad renal en función del sexo y del genotipo, clasificar los pacientes según la pérdida anual de función renal (progresión renal rápida, moderada y lenta), identificar biomarcadores clínicos de pronóstico, y evidenciar la alta variabilidad fenotípica intrafamiliar. El estudio para la identificación de variantes génicas en homocigosis asociadas a los fenotipos más extremos (progresión renal rápida y lenta) se realizó con los datos obtenidos de la secuenciación masiva del exoma de los 30 pacientes de la cohorte. Con esta estrategia, se identificaron 45 variantes génicas. De entre estas, por la función fisiológica de los genes donde se localizan, destacan la rs11207827 (en el gen PATJ) y la rs1050171 (en el gen EGFR). Para determinar los factores epigenéticos implicados en la fisiopatología de la HFHNC y en su progresión, se utilizaron los urinary exosome-like vesicles (uEVs) como fuente no invasiva de información de procesos celulares renales. Mediante microarrays, se analizó el perfil de expresión de los miRNAs contenidos en los uEVs de los 20 pacientes que mantenían los riñones nativos funcionales, identificándose 24 miRNAs diferencialmente expresados en el total de pacientes respecto al grupo control, y 43 en el subconjunto de pacientes homocigotos para la mutación p.G20D en CLDN19. La comparación de pacientes de ambos sexos mostro únicamente la infraexpresión del miR-1915-5p en aquellos de sexo masculino. Finalmente, se identificaron 4 miRNAs diferencialmente expresados en los pacientes con progresión renal moderada, en comparación con el de progresión lenta, y 8 en el subgrupo de pacientes homocigotos para la mutación p.G20D en CLDN19. La biología de sistemas y el uso de redes neuronales de inteligencia artificial han permitido asociar los resultados obtenidos con procesos biológicos cruciales en la fisiopatología de la HFHNC, como la fibrosis renal y el transporte de calcio y magnesio, entre otros. Este trabajo ha permitido incrementar el conocimiento de la fisiopatología de la HFHNC y determinar factores clínicos, genéticos y epigenéticos implicados en la disfunción renal. Desde el punto de vista traslacional, se han identificado nuevos posibles biomarcadores pronósticos de la HFHNC, y dianas terapéuticas que podrían permitir modular la severidad de la enfermedad y, en el mejor de los casos, curarla.Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive tubulopathy caused by mutations in either CLDN16 or CLDN19 genes, which encode for claudin-16 and -19, respectively, expressed in the thick ascending loop of Henle, and involved in paracellular ion transport. This disease is characterized by urinary loss of calcium and magnesium, bilateral nephrocalcinosis, and inexorable progression of chronic renal disease leading to renal failure. Besides, most patients with CLDN19 mutations also develop ocular anomalies, as it is expressed in the retinal epithelial cells. There is great phenotypic variability among patients, even in those who share the c.59G>A; p.G20D mutation in CLDN19 (Hispanic mutation), and also among members of the same family. This phenomenon suggests that, beyond the mutation causing the disease, other molecular processes could determine the clinical progression of patients. Under this hypothesis, this work has focused on the study of genetic and epigenetic factors that could modulate renal disease progression. For this purpose, clinical data, urine and blood from 30 patients affected by FHHNC and 6 control individuals were collected from around Spain, being them the majority of patients diagnosed of FHHNC in this territory. The analysis of the clinical data allowed evaluating the differences in the evolution of the renal disease according to sex and genotype, classifying patients according to the annual decline of renal function (fast, moderate and slow renal progression), identifying clinical biomarkers of prognosis, and evidencing the high intrafamilial phenotypic variability. The study for the identification of gene variants in homozygosis associated with both extreme phenotypes (fast and slow renal progression) was carried out with data obtained from exome sequencing of the 30 patients included in the cohort. With this strategy, 45 gene variants were identified. Among these, due to the physiological function of the genes where they are located, the rs11207827 (in the PATJ gene) and the rs1050171 (in the EGFR gene) were highlighted. To determine the epigenetic factors involved in FHHNC physiopathology and disease progression, the urinary exosome-like vesicles (uEVs) were used as a non-invasive source of information on renal cellular processes. Microarray technique was used to analyze the expression pattern of miRNAs contained in uEVs of the 20 patients who maintained functional native kidneys. Twenty-four miRNAs were identified differentially expressed in all patients when compared with the control group, and 43 in the subset of patients homozygous for the p.G20D mutation in CLDN19. The comparison of patients of both sexes showed only the under-expression of the miR-1915-5p in males. Finally, 4 miRNAs were differentially expressed in patients with moderate to slow progression of renal disease, and 8 within the subgroup of patients homozygous for the p.G20D mutation in CLDN19. Systems biology and the use of artificial neuronal networks have allowed to associate the results obtained with crucial biological processes in FHHNC physiopathology such as renal fibrosis and calcium and magnesium transport, among others. This work, through two complementary strategies, has allowed increasing the knowledge of the physiopathology of FHHNC and, for the first time, determining clinical, genetic and epigenetic factors involved in renal disease progression. From a translational perspective, this thesis has identified new possible prognostic biomarkers of FHHNC as well as novel therapeutic targets that could allow modulating the severity of the disease and, in the best case, to cure it

Subcutaneous anti-COVID-19 hyperimmune immunoglobulin for prevention of disease in asymptomatic individuals with SARS-CoV-2 infection: a double-blind, placebo-controlled, randomised clinical trialResearch in context

Summary: Background: Anti-COVID-19 hyperimmune immunoglobulin (hIG) can provide standardized and controlled antibody content. Data from controlled clinical trials using hIG for the prevention or treatment of COVID-19 outpatients have not been reported. We assessed the safety and efficacy of subcutaneous anti-COVID-19 hyperimmune immunoglobulin 20% (C19-IG20%) compared to placebo in preventing development of symptomatic COVID-19 in asymptomatic individuals with SARS-CoV-2 infection. Methods: We did a multicentre, randomized, double-blind, placebo-controlled trial, in asymptomatic unvaccinated adults (≥18 years of age) with confirmed SARS-CoV-2 infection within 5 days between April 28 and December 27, 2021. Participants were randomly assigned (1:1:1) to receive a blinded subcutaneous infusion of 10 mL with 1 g or 2 g of C19-IG20%, or an equivalent volume of saline as placebo. The primary endpoint was the proportion of participants who remained asymptomatic through day 14 after infusion. Secondary endpoints included the proportion of individuals who required oxygen supplementation, any medically attended visit, hospitalisation, or ICU, and viral load reduction and viral clearance in nasopharyngeal swabs. Safety was assessed as the proportion of patients with adverse events. The trial was terminated early due to a lack of potential benefit in the target population in a planned interim analysis conducted in December 2021. ClinicalTrials.gov registry: NCT04847141. Findings: 461 individuals (mean age 39.6 years [SD 12.8]) were randomized and received the intervention within a mean of 3.1 (SD 1.27) days from a positive SARS-CoV-2 test. In the prespecified modified intention-to-treat analysis that included only participants who received a subcutaneous infusion, the primary outcome occurred in 59.9% (91/152) of participants receiving 1 g C19-IG20%, 64.7% (99/153) receiving 2 g, and 63.5% (99/156) receiving placebo (difference in proportions 1 g C19-IG20% vs. placebo, −3.6%; 95% CI -14.6% to 7.3%, p = 0.53; 2 g C19-IG20% vs placebo, 1.1%; −9.6% to 11.9%, p = 0.85). None of the secondary clinical efficacy endpoints or virological endpoints were significantly different between study groups. Adverse event rate was similar between groups, and no severe or life-threatening adverse events related to investigational product infusion were reported. Interpretation: Our findings suggested that administration of subcutaneous human hyperimmune immunoglobulin C19-IG20% to asymptomatic individuals with SARS-CoV-2 infection was safe but did not prevent development of symptomatic COVID-19. Funding: Grifols