Isolation and characterization of exosome-enriched urinary extracellular vesicles from Dent's disease type 1 Spanish patients

Enfermedad de Dent; Exosomas; Vesículas extracelularesDent's disease; Exosomes; Extracelular vesiclesMalaltia de Dent; Exosomes; Vesícules extracel·lularsAntecedentes y objetivo La enfermedad de Dent tipo 1 (DD1) es una enfermedad hereditaria rara ligada al cromosoma X causada por mutaciones en el CLCN5 que se caracteriza principalmente por una disfunción del túbulo proximal, hipercalciuria, nefrolitiasis o nefrocalcinosis, enfermedad renal crónica progresiva y proteinuria de bajo peso molecular, rasgo distintivo de la enfermedad. En la actualidad no existe un tratamiento curativo específico, únicamente sintomático, y no previene la progresión de la enfermedad. En este estudio hemos aislado y caracterizado las vesículas extracelulares urinarias (uEV) enriquecidas en exosomas que nos permitirán identificar biomarcadores asociados a la progresión de DD1 y ayudarán a una mejor comprensión de las bases fisiopatológicas. Materiales y métodos A través de una convocatoria nacional de la Sociedad Española de Nefrología (SEN) y la Sociedad Española de Nefrología Pediátrica (AENP), se obtuvieron orinas de pacientes y controles de distintos hospitales españoles, las cuales se procesaron para obtener los uEV. Los datos de estos pacientes fueron proporcionados por los respectivos nefrólogos o extraídos del registro RENALTUBE. Los uEV se aislaron mediante ultracentrifugación, fueron caracterizados morfológicamente y se extrajo su contenido de proteína y micro-ARN. Resultados Se seleccionó a 25 pacientes y 10 controles, de los cuales se procesaron las orinas para aislar los uEV. Nuestros resultados mostraron que la concentración relativa de uEV/ml era menor en los pacientes que en los controles (0,26 × 106 vs. 1,19 × 106 uEV/ml; p < 0,01). Además, se vio que los uEV de los pacientes eran significativamente más grandes que los de los sujetos control (diámetro medio: 187,8 vs. 143,6 nm; p < 0,01). Por último, nuestros datos demostraron que se había extraído correctamente el ARN tanto de los exosomas de pacientes como de los controles. Conclusiones En este trabajo describimos el aislamiento y caracterización de uEV de pacientes con DD 1 y controles sanos, útiles para el posterior estudio de moléculas cargo diferencialmente expresadas en esta enfermedad.Background and objectives Dent's disease type 1 (DD1) is a rare X-linked hereditary pathology caused by CLCN5 mutations that is characterized mainly by proximal tubule dysfunction, hypercalciuria, nephrolithiasis/nephrocalcinosis, progressive chronic kidney disease, and low-weight proteinuria, the molecular hallmark of the disease. Currently, there is no specific curative treatment, only symptomatic and does not prevent the progression of the disease. In this study we have isolated and characterized urinary extracellular vesicles (uEVs) enriched in exosomes that will allow us to identify biomarkers associated with DD1 progression and a better understanding of the pathophysiological bases of the disease. Materials and methods Through a national call from the Spanish Society of Nephrology (SEN) and the Spanish Society of Pediatric Nephrology (AENP), urine samples were obtained from patients and controls from different Spanish hospitals, which were processed to obtain the uEVs. The data of these patients were provided by the respective nephrologists and/or extracted from the RENALTUBE registry. The uEVs were isolated by ultracentrifugation, morphologically characterized and their protein and microRNA content extracted. Results Twenty-five patients and 10 controls were recruited, from which the urine was processed to isolate the uEVs. Our results showed that the relative concentration of uEVs/ml is lower in patients compared to controls (0.26 × 106 vs. 1.19 × 106 uEVs/ml, P < 0.01). In addition, the uEVs of the patients were found to be significantly larger than those of the control subjects (mean diameter: 187.8 vs. 143.6 nm, P < 0.01). Finally, our data demonstrated that RNA had been correctly extracted from both patient and control exosomes. Conclusions In this work we describe the isolation and characterization of uEVs from patients with DD1 and healthy controls, that shall be useful for the subsequent study of differentially expressed cargo molecules in this pathology.Este trabajo ha sido financiado principalmente por la fundación SENEFRO (SEN2019 a AM), por ASDENT y por subvenciones del Ministerio de Ciencia e Innovación (SAF201789989 a AM) y de la Red de Investigación Renal REDinREN (12/0021/0013). El Grupo de Fisiopatología Renal tiene la Mención de Calidad de la Generalitat de Cataluña (2017 SGR)

Discovery of putative prognostic and therapeutic miRNA in uEVs of Dent's Disease 1 patients and characterisation of cellular models of the disease

Dent disease 1 (DD1) is a rare renal tubulopathy caused by CLCN5 mutations and characterized by low molecular weight proteinuria, variable hypercalciuria, nephrocalcinosis and/or nephrolithiasis and progression to kidney failure. The underlying mechanisms linking ClC-5 loss-of-function and endocytosis impairment in the renal proximal tubule (and other DD1 phenotypes) remain unknown. In this thesis we have followed three approaches to identify altered pathways by ClC-5 mutations: (1) conduct a European survey to analyse the prevalence and DD1 clinical features, (2) study miRNA expression profiles from DD1 patients’ urinary exosome-like vesicles (uEVs) to get insight into DD1 pathophysiological mechanisms and (3) characterisation of a DD1 cell model. The European survey showed that DD1 has a variable presentation. Our study of uEVs miRNA identified new pathophysiological pathways, which may lead to identify putative diagnostic and prognostic biomarkers. Finally, our cell model with different mutations provides a valuable prototype for additional investigation of impaired pathways.La malaltia de Dent 1 (DD1) és una tubulopatia renal rara causada per mutacions en el gen CLCN5 i caracteritzada per proteinuria de baix pes molecular, hipercalciuria, nefrocalcinosi i/o litiasis renals així com progrés a insuficiència renal. Els mecanismes que causen la pèrdua de funció de ClC-5 i el defecte en l’endocitosi en el túbul proximal (entre d’altres fenotips de DD1) no es coneixen. En aquesta Tesi hem desenvolupat tres aproximacions per identificar vies alterades per mutacions en ClC-5. (1) hem fet una enquesta europea per analitzar la prevalença i les característiques clíniques de DD1, (2) hem estudiat l’expressió de miRNA en vesícules exosome-like urinàries (uEVs) per entendre els mecanismes fisiopatològics de la malaltia i (3) hem caracteritzat un model cel·lular de DD1. L’enquesta europea mostrà que DD1 té una presentació variable. El nostre estudi de miRNA en uEVs va permetre identificar nous mecanismes fisiopatològics que poden ser potencials biomarcadors diagnòstics i pronòstics de DD1. Finalment, el nostre model cel·lular amb diferents mutacions provà representar un prototip vàlid per investigacions addicionals del mecanismes desregulats

Treatment with sotrovimab for SARS-CoV-2 infection in a cohort of high-risk kidney transplant recipients

Background Sotrovimab is a neutralizing monoclonal antibody (mAb) that seems to remain active against recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. The evidence on its use in kidney transplant (KT) recipients, however, is limited. Methods We performed a multicenter, retrospective cohort study of 82 KT patients with SARS-CoV-2 infection {coronavirus disease 2019 [COVID-19]} treated with sotrovimab. Results Median age was 63 years. Diabetes was present in 43.9% of patients, and obesity in 32.9% of patients; 48.8% of patients had an estimated glomerular filtration rate under 30 mL/minute/1.73 m2. Additional anti–COVID-19 therapies were administered to 56 patients, especially intravenous steroids (65.9%). Sotrovimab was administered early (<5 days from the onset of the symptoms) in 46 patients (56%). Early-treated patients showed less likely progression to severe COVID-19 than those treated later, represented as a lower need for ventilator support (2.2% vs 36.1%; P < .001) or intensive care admission (2.2% vs 25%; P = .002) and COVID-19–related mortality (2.2% vs 16.7%; P = .020). In the multivariable analysis, controlling for baseline risk factors to severe COVID-19 in KT recipients, early use of sotrovimab remained as a protective factor for a composite outcome, including need for ventilator support, intensive care, and COVID-19–related mortality. No anaphylactic reactions, acute rejection episodes, impaired kidney function events, or non-kidney side effects related to sotrovimab were observed. Conclusions Sotrovimab had an excellent safety profile, even in high-comorbidity patients and advanced chronic kidney disease stages. Earlier administration could prevent progression to severe disease, while clinical outcomes were poor in patients treated later. Larger controlled studies enrolling KT recipients are warranted to elucidate the true efficacy of monoclonal antibody therapies

Novel Dent disease 1 cellular models reveal biological processes underlying ClC-5 loss-of-function

Dent disease 1 (DD1) is a rare X-linked renal proximal tubulopathy characterized by low molecular weight proteinuria and variable degree of hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressing to chronic kidney disease. Although mutations in the electrogenic Cl-/H+ antiporter ClC-5, which impair endocytic uptake in proximal tubule cells, cause the disease, there is poor genotype-phenotype correlation and their contribution to proximal tubule dysfunction remains unclear. To further discover the mechanisms linking ClC-5 loss-of-function to proximal tubule dysfunction, we have generated novel DD1 cellular models depleted of ClC-5 and carrying ClC-5 mutants p.(Val523del), p.(Glu527Asp) and p.(Ile524Lys) using the human proximal tubule-derived RPTEC/TERT1 cell line. Our DD1 cellular models exhibit impaired albumin endocytosis, increased substrate adhesion and decreased collective migration, correlating with a less differentiated epithelial phenotype. Despite sharing functional features, these DD1 cell models exhibit different gene expression profiles, being p.(Val523del) ClC-5 the mutation showing the largest differences. Gene set enrichment analysis pointed to kidney development, anion homeostasis, organic acid transport, extracellular matrix organization and cell-migration biological processes as the most likely involved in DD1 pathophysiology. In conclusion, our results revealed the pathways linking ClC-5 mutations with tubular dysfunction and, importantly, provide new cellular models to further study DD1 pathophysiology.This work was supported in part by Asdent Patients Association and grants from Ministerio de Ciencia e Innovación (SAF201459945-R and SAF201789989-R to A.M.), the Fundación Senefro (SEN2019 to A.M.) and Red de Investigación Renal REDinREN (12/0021/0013). A.M. group holds the Quality Mention from the Generalitat de Catalunya (2017 SGR)

Early hypertransaminasemia after kidney transplantation: significance and evolution according to donor type

Early hypertransaminasemia after kidney transplantation (KT) is frequent. It has been associated with the crosstalk produced between the liver and the kidney in ischemia-reperfusion situations. However, the influence of the donor type has not been evaluated. We present a retrospective study analyzing the increase in serum aspartate aminotransferase/alanine aminotransferase (AST/ALT) during the first three months post-KT in 151 recipients who received thymoglobulin as induction therapy, either from brain-death donors (DBD, n = 75), controlled circulatory death donors (cDCD, n = 33), or uncontrolled DCD (uDCD, n = 43). Eighty-five KT recipients from DBD who received basiliximab were included as controls. From KT recipients who received thymoglobulin, 33.6/43.4% presented with an increase in AST/ALT at 72 h post-KT, respectively. Regarding donor type, the percentage of recipients who experienced 72 h post-KT hypertransaminasemia was higher in uDCD group (65.1/83.7% vs. 20.3/26% in DBD and 20.7/27.6% in cDCD, p < 0.001). Within the control group, 9.4/12.9% of patients presented with AST/ALT elevation. One month after transplant, AST/ALT values returned to baseline in all groups. The multivariate analysis showed that uDCD recipients had 6- to 12-fold higher risk of developing early post-KT hypertransaminasemia. Early post-KT hypertransaminasemia is a frequent and transient event related to the kidney donor type, being more frequent in uDCD recipients

Evolución de las causas de pérdida del injerto en trasplante renal durante 40 años (1979-2019)

Article disponible en anglès: http://hdl.handle.net/10230/60080Introducción: La mejoría en la supervivencia del receptor y del injerto renal sufre un proceso de deceleración. La tasa de pérdida del injerto a medio y largo plazo permanece estable desde hace 25 años. Es fundamental conocer las causas de pérdida del injerto y los factores relacionados, así como identificar si se han producido cambios en las causas de pérdida del injerto en los últimos años. El objetivo del presente estudio fue evaluar las causas de pérdida del injerto según fallecimiento del receptor o pérdida del injerto con vuelta a diálisis/retrasplante, y analizar las causas específicas de pérdida del injerto en 2 épocas (1979-1999 y 2000-2019) para identificar cambios en el perfil de los injertos perdidos. Pacientes y métodos: Estudio retrospectivo de todos los trasplantes renales (TR) realizados en el Hospital del Mar (Barcelona) entre mayo-1979 y diciembre-2019. Consideramos pérdida del injerto el fallecimiento del paciente con injerto funcionante o el re-inicio de diálisis o retrasplante. Revisamos las causas de pérdida mediante información clínica e histológica, y analizamos los resultados en 2 periodos (1979-1999 y 2000-2019). Resultados: De los 1.522 TR realizados, 722 (47,5%) perdieron el injerto renal con una mediana de seguimiento hasta la pérdida de 56 (RIQ: 8-123) meses. De estas pérdidas, 483 (66,9%) se produjeron por fracaso del injerto y 239 (33,1%) por fallecimiento del receptor con injerto funcionante. Las causas del fallecimiento fueron las cardiovasculares (25,1%), las neoplasias (25,1%) y las infecciones (21,8%), de forma similar en ambas épocas de trasplante. Solo el éxitus de causa desconocida ha disminuido en la época más reciente. La principal causa de vuelta a diálisis/retrasplante fue la disfunción del injerto (75%), siendo el rechazo mediado por anticuerpos y la fibrosis intersticial/atrofia tubular los hallazgos histológicos más frecuentes (15,9 y 12,6%). Del total de pérdidas, 213 fueron precoces (29,5%). La trombosis vascular fue la causa más frecuente de pérdida precoz (<1 año posTR) entre 2000 y 2019 (46,7%), mientras que en el periodo 1979-1999 lo fue el rechazo celular (31,3%). Las causas de pérdida del injerto tardías fueron similares en ambas épocas. Conclusiones: Los pacientes fallecen de causas cardiovasculares y neoplasias, y esto no ha cambiado con los años. La trombosis vascular emerge como causa frecuente de pérdida precoz del injerto en la época actual. La identificación adecuada de las causas de pérdida del injerto es fundamental para mejorar los resultados postrasplante

Outcomes of frail patients while waiting for kidney transplantation: differences between physical frailty phenotype and FRAIL scale

Frailty is associated with poorer outcomes among patients waiting for kidney transplantation (KT). Several different tools to measure frailty have been used; however, their predictive value is unknown. This is a prospective longitudinal study of 449 KT candidates evaluated for frailty by the Physical Frailty Phenotype (PFP) and the FRAIL scale. During the study period, 296 patients received a KT, while 153 remained listed. Patients who did not get receive a transplant were more frequently frail according to PFP (16.3 vs. 7.4%, p = 0.013). Robust patients had fewer hospital admissions during the 1st year after listing (20.8% if PFP = 0 vs. 43.4% if ≥1, and 27.1% if FRAIL = 0 vs. 48.9% if ≥1) and fewer cardiovascular events (than FRAIL ≥ 1) or major infectious events (than PFP ≥ 1). According to PFP, scoring 1 point had an impact on patient survival and chance of transplantation in the univariate analysis. The multivariable analysis corroborated the result, as candidates with PFP ≥ 3 had less likelihood of transplantation (HR 0.45 [0.26-0.77]). The FRAIL scale did not associate with any of these outcomes. In KT candidates, pre-frailty and frailty according to both the PFP and the FRAIL scale were associated with poorer results while listed. The PFP detected that frail patients were less likely to receive a KT, while the FRAIL scale did not

Evolution of kidney allograft loss causes over 40 years (1979-2019)

Article disponible en castellà: http://hdl.handle.net/10230/60085Introduction: The improvement of kidney allograft recipient and graft survival showed a decrease over the last 40 years. Long-term graft loss rate remained stable during a 25-year time span. Knowing the changing causes and the risk factors associated with graft loss requires special attention. The present study aimed to assess the causes of graft loss and kidney allograft recipient death. Also, we aimed to compare two different periods (1979-1999 and 2000-2019) to identify changes in the characteristics of the failed allografts and recipient and donors profile. Methods and patients: We performed a single-center cohort study. We included all the kidney transplant recipients at the Hospital del Mar (Barcelona) between May 1979 and December 2019. Graft loss was defined as recipient death with functioning graft and as loss of graft function (return to dialysis or retransplantation). We assessed the causes of graft loss using clinical and histological information. We also analyzed the results of the two different transplant periods (1979-1999 and 2000-2019). Results: Between 1979 and 2019, 1522 transplants were performed. The median follow-up time was 56 (IQR 8-123) months. During follow-up, 722 (47.5%) grafts were lost: 483 (66.9%) due to graft failure and 239 (33.1%) due to death with functioning graft. The main causes of death were cardiovascular (25.1%), neoplasms (25.1%), and infectious diseases (21.8%). These causes were stable between the two periods of time. Only the unknown cause of death has decreased in the last period. The main cause of graft failure (loss of graft function) was the allograft chronic dysfunction (75%). When histologic information was available, antibody-mediated rejection (ABMR) and interstitial fibrosis/tubular atrophy (IF/TA) were the most frequent specific causes (15.9% and 12.6%). Of the graft failures, 213 (29.5%) were early (<1 year of transplantation). Vascular thrombosis was the main cause of early graft failure in the second period (2000-2019) (46.7%) and T-cell-mediated rejection (TCMR) was the main cause (31.3%) in the first period (1979-1999). The causes of late graft loss were similar between the two periods. Conclusions: The causes of kidney allograft recipient death are still due to cardiovascular and malignant diseases. Vascular thrombosis has emerged as a frequent cause of early graft loss in the most recent years. The evaluation of the causes of graft loss is necessary to improve kidney transplantation outcomes