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)

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

Increased mortality after kidney transplantation in mildly frail recipients

Physical Frailty Phenotype (PFP) is the most used frailty instrument among kidney transplant recipients, classifying patients as pre-frail if they have 1-2 criteria and as frail if they have ≥3. However, different definitions of robustness have been used among renal patients, including only those who have 0 criteria, or those with 0-1 criteria. Our aim was to determine the impact of one PFP criterion on transplant outcomes. We undertook a retrospective study of 296 kidney transplant recipients who had been evaluated for frailty by PFP at the time of evaluating for transplantation. Only 30.4% of patients had 0 criteria, and an additional 42.9% showed one PFP criterion. As PFP score increased, a higher percentage of women and cerebrovascular disease were found. Recipients with 0-1 criteria had lower 1-year mortality after transplant than those with ≥2 (1.8% vs 10.1%), but this difference was already present when we only considered those who scored 0 (mortality 1.1%) and 1 (mortality 2.4%) separately. The multivariable analysis confirmed that one PFP criterion was associated to a higher risk of patient death after kidney transplantation [hazard ratio 3.52 (95% confidence interval 1.03-15.9)]. Listed kidney transplant candidates frequently show only one PFP frailty criterion. This has an independent impact on patient survival after transplantation

Validation of a survival benefit estimator tool in a cohort of European kidney transplant recipients

Producción CientíficaPre-transplant prognostic scores help to optimize donor/recipient allocation and to minimize organ discard rates. Since most of these scores come from the US, direct application in non-US populations is not advisable. The Survival Benefit Estimator (SBE), built upon the Estimated Post-Transplant Survival (EPTS) and the Kidney Donor Profile Index (KDPI), has not been externally validated. We aimed to examine SBE in a cohort of Spanish kidney transplant recipients. We designed a retrospective cohortbased study of deceased-donor kidney transplants carried out in two different Spanish hospitals. Unadjusted and adjusted Cox models were applied for patient survival. Predictive models were compared using Harrell’s C statistics. SBE, EPTS and KDPI were independently associated with patient survival (p ≤ 0.01 in all models). Model discrimination measured with Harrell’s C statistics ranged from 0.57 (KDPI) to 0.69 (SBE) and 0.71 (EPTS). After adjustment, SBE presented similar calibration and discrimination power to that of EPTS. SBE tended to underestimate actual survival, mainly among high EPTS recipients/high KDPI donors. SBE performed acceptably well at discriminating posttransplant survival in a cohort of Spanish deceased-donor kidney transplant recipients, although its use as the main allocation guide, especially for high KDPI donors or high EPTS recipients requires further testing.Rio Hortega contract (ISCIII-11453)Fondo de Investigaciones Sanitarias - Fondo Europeo de Desarrollo Regional (project PI16/0617)Redinren (project RD16/0009/001

Recurrence of iga nephropathy after kidney transplantation in adults

Background and objectives: In patients with kidney failure due to IgA nephropathy, IgA deposits can recur in a subsequent kidney transplant. The incidence, effect, and risk factors of IgA nephropathy recurrence is unclear, because most studies have been single center and sample sizes are relatively small. Design, setting, participants, & measurements: We performed a multicenter, international, retrospective study to determine the incidence, risk factors, and treatment response of recurrent IgA nephropathy after kidney transplantation. Data were collected from all consecutive patients with biopsy-proven IgA nephropathy transplanted between 2005 and 2015, across 16 “The Post-Transplant Glomerular Disease” study centers in Europe, North America, and South America. Results: Out of 504 transplant recipients with IgA nephropathy, recurrent IgA deposits were identified by kidney biopsy in 82 patients; cumulative incidence of recurrence was 23% at 15 years (95% confidence interval, 14 to 34). Multivariable Cox regression revealed a higher risk for recurrence of IgA deposits in patients with a pre-emptive kidney transplant (hazard ratio, 3.45; 95% confidence interval, 1.31 to 9.17) and in patients with preformed donorspecific antibodies (hazardratio, 2.59; 95%confidence interval, 1.09 to 6.19).Afterkidneytransplantation,development of de novo donor-specific antibodies was associated with subsequent higher risk of recurrence of IgA nephropathy (hazard ratio, 6.65; 95% confidence interval, 3.33 to 13.27). Immunosuppressive regimen was not associated with recurrent IgA nephropathy in multivariable analysis, including steroid use. Graft loss was higher in patients with recurrence of IgA nephropathy compared with patients without (hazard ratio, 3.69; 95% confidence interval, 2.04 to 6.66), resulting in 32% (95% confidence interval, 50 to 82) graft loss at 8 years after diagnosis of recurrence. Conclusions: In our international cohort, cumulative risk of IgA nephropathy recurrence increased after transplant and was associated with a 3.7-fold greater risk of graft loss

Recovery of dialysis patients with COVID-19 : health outcomes 3 months after diagnosis in ERACODA

Background. Coronavirus disease 2019 (COVID-19)-related short-term mortality is high in dialysis patients, but longer-term outcomes are largely unknown. We therefore assessed patient recovery in a large cohort of dialysis patients 3 months after their COVID-19 diagnosis. Methods. We analyzed data on dialysis patients diagnosed with COVID-19 from 1 February 2020 to 31 March 2021 from the European Renal Association COVID-19 Database (ERACODA). The outcomes studied were patient survival, residence and functional and mental health status (estimated by their treating physician) 3 months after COVID-19 diagnosis. Complete follow-up data were available for 854 surviving patients. Patient characteristics associated with recovery were analyzed using logistic regression. Results. In 2449 hemodialysis patients (mean ± SD age 67.5 ± 14.4 years, 62% male), survival probabilities at 3 months after COVID-19 diagnosis were 90% for nonhospitalized patients (n = 1087), 73% for patients admitted to the hospital but not to an intensive care unit (ICU) (n = 1165) and 40% for those admitted to an ICU (n = 197). Patient survival hardly decreased between 28 days and 3 months after COVID-19 diagnosis. At 3 months, 87% functioned at their pre-existent functional and 94% at their pre-existent mental level. Only few of the surviving patients were still admitted to the hospital (0.8-6.3%) or a nursing home (∼5%). A higher age and frailty score at presentation and ICU admission were associated with worse functional outcome. Conclusions. Mortality between 28 days and 3 months after COVID-19 diagnosis was low and the majority of patients who survived COVID-19 recovered to their pre-existent functional and mental health level at 3 months after diagnosis

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

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

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: 25 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 uEVs/mL 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 nm 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 Dent 1 disease and healthy controls, that shall be useful for the subsequent study of differentially expressed cargo molecules in this pathology. Resumen: Antecedentes y objetivo: La enfermedad de Dent tipo 1 (DD1) es una patología hereditaria rara ligada al cromosoma X causado por mutaciones en el CLCN5 que se caracteriza principalmente por una disfunción del túbulo proximal, hipercalciuria, nefrolitiasis/nefrocalcinosis, enfermedad renal crónica progresiva y proteinuria de bajo peso molecular, rasgo distintivo de la enfermedad. Actualmente, 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 (uEVs) enriquecidas en exosomas que nos permitirán identificar biomarcadores asociados a la progresión de DD1 y una mejor comprensión de las bases fisiopatológicas de la misma. 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 uEVS. Los datos de estos pacientes fueron proporcionados por los respectivos nefrólogos y/o extraídos del registro RENALTUBE. Los uEVs se aislaron mediante ultracentrifugación, fueron caracterizados morfológicamente y se extrajo su contenido de proteína y microRNA. Resultados: Se reclutaron 25 pacientes y 10 controles, de los cuales se procesaron las orinas para aislar los uEVs. Nuestros resultados mostraron que la concentración relativa de uEVs/mL es menor en pacientes comparado con controles (0,26 × 106 uEVs/mL vs 1,19 × 106 uEVs/mL, p < 0.01). Además, se vio que los uEVs de los pacientes eran significativamente más grandes que los de los sujetos control (diámetro medio: 187,8 nm vs 143,6 nm, p < 0.01). Finalmente, nuestros datos demostraron que se había extraído correctamente RNA tanto de los exosomas de pacientes como de controles. Conclusiones: En este trabajo describimos el aislamiento y caracterización de uEVs de pacientes de la enfermedad de Dent 1 y controles sanos, útiles para el posterior estudio de moléculas cargo diferencialmente expresadas en esta patología

Impact of recurrent acute kidney injury on patient outcomes

BACKGROUND/AIMS: Recurrent acute kidney injury (AKI) is common among patients after a first hospitalized AKI. However, little is known about the prognosis of recurrent AKI episodes in chronic kidney disease (CKD) development, cardiovascular events and mortality. METHODS: A retrospective study included patients admitted to our Hospital from 2000 to 2010. AKI was defined according to the Acute Dialysis Quality Initiative criteria. In the follow-up period after the first AKI episode, clinical, laboratory data and the number of repeated AKI episodes, etiology and severity were recorded. RESULTS: Among the 359 AKI survivor patients included, 250 new AKI episodes were observed in 122 patients (34%). Variables independently associated to new episodes were: type 2 DM [OR 1.2, 95%CI 1.2-3.8, p=0.001], ischemic heart disease [OR 1.9; 95%CI 1.1-3.6, p=0.012], and SCr at the first AKI event>2,6 mg/dl [OR 1.2; 95%CI 1.03-1.42, p=0.02]. Development of CKD during four years follow-up was more frequent in patients with recurrent AKI, HR [2.2 (95% CI: 1.09-4.3, p=0.003)] and 44% of recurrent AKI patients who developed CKD occurred during the first 6 months after the initial event. Cardiovascular events were more frequent among patients with recurrent AKI patients than in those with one AKI episode (47.2% vs 24%, p=0.001). Mortality at 4 years was higher in the patient subgroup with several episodes of AKI as compared with those with a single episode [HR: 4.5 (95% CI 2.7-7.5) p<0.001]. CONCLUSION: Episodes of recurrent AKI have a high potential to be associated with relevant complications such as cardiovascular events, mortality and CKD development