Javier Juega

Technical Characteristics, Optimization and Outcomes of Ultrasound Guided Biopsy Performed on Kidney Transplant Patients

REDinREN. REMAR. Materials and Methods From January 2014 to August 2016, 183 US-guided biopsies were performed in our institution on renal transplanted patients. Over that period We decided to optimize our biopsy protocol, switching from the initial 24 hours hospitalization and observation period of every patient undergoing a graft biopsy to an outpatient protocol of 6 hours observation and discharge after blood count control. Furthermore, we evolved from an initially shared technique made by radiologists and nephrologist together to the current US guided biopsy performed by the nephrologist alone. On the other hand we decided to change from the 14 G biopsy needle to the more suitable 16 G needle over the same period of time, being used on the outcome and the nephrologist alone protocols. We retrospectively analyzed the results of those changes and compared outcomes of each group Results 183 renal graft biopsies performed. 79,8% by radiologist and nephrologist together. 20,2% by nephrologist alone. Outpatient protocol with 6 hours observation performed on 51,4% of patients. 48,6% underwent programed admission and discharge after 24 hours observation. Automatic biopsy gun 14 G were used in 43,2%, 16 G in 37,7%, 18 G in 6%. 13,1% needle information not recorded. Mean age was 54 ± 14, 66,7% male. Mean needle passes 2 ± 1, mean number of useful samples 1,5 ±0,5. Mean pre-biopsy hemoglobin 11,5 ± 1,8 gr/dl, post-biopsy 10,9 ± 1,75 gr/dl, and mean Hb change was 0,65 ± 0,62. Mean obtained glomeruli was 18 ± 11. The 14 G biopsy gun was mainly used for the inpatient protocol ( 52%) and for the nephrologist and radiologist shared technique ( 54,8%), while 16 G biopsy gun was mainly used in outpatient cases ( 47%) and in all nephrologist independent technique cases. The overall rate of complications was 7,7%. It was higher in the shared technique cases vs. Nephrologist independent cases ( 8,2 vs 5,4%). There were no differences between complications observed in the inpatient vs. outpatient cases ( 7,8 vs. 7,4%). There were more complications observed in those biopsies using 14 G compared to 16 G ( 11,2 vs. 4,3%) Only one case ( 0,3%) required intravascular segmental embolization of transplanted kidney due to severe active bleeding after biopsy. There were no nephrectomies or deaths during the study period. Conclusion We observed fewer complications with appropriate sample obtainment using 16 G biopsy needle compared with 14 G. We found no differences in complications rate between inpatient vs. outpatient with 6 hours of observation protocol. We found less complications when biopsy performed by nephrologist alone compared with shared technique, although when comparing only cases in which 16 G needle was used, complication rates were similar. We believe US-guided renal graft biopsy using 16 G needle, with only 6 hours of observation after procedure and performed by nephrologist alone is safe,efficient, cost-saving and optimizes the renal graft biopsy process.

Urinary miRNA as a Potential Biomarker in Kidney Transplantation

Introduction and Aims Kidney transplantation is the best available treatment for patients with end stage chronic kidney disease. The diagnosis of the pathology of the kidney graft relies on the determination of the creatinine, estimated glomerular filtration rate and proteinuria. These parameters are altered after the onset of renal injury and they are not able to discern between various pathologies. The kidney biopsy, an invasive and not extent of complications procedure, remains essential for the diagnosis of the renal pathology. Hence the importance of finding parameters that allow an early diagnosis of the pathology involved in graft dysfunction. Urine is ideal for the study of possible biomarkers, its collection is easy, non-invasive and repeatable. Our aim was to analyse the miRNA content of urinary extracellular vesicles of the transplanted patients and define differential patterns of expression of miRNA specific to the different pathologies of the transplanted kidney. Methods Kidney transplanted patients with a kidney biopsy performed for clinical indication were selected. The biopsies were assessed according to Banff classification. From all biopsies those exhibiting acute cellular rejection (ACR), interstitial fibrosis and tubular atrophy (IFTA) and calcineurin inhibitors toxicity (CIT) were selected. Kidney transplanted patients with normal function (defined as eGFR CKD-EPI >90ml/min/1,72m2) and without proteinuria were defined as control group. First morning void urine was collected from patients. After low speed centrifugation to eliminate cell debris, Urine EVs were obtained by Size exclusion chromatography after urine concentration. EV-contained fractions selected as CD63 and CD9 positive fractions were pooled. Then, total RNA was extracted using mirCURY kit and high-throughput sequencing was done. Taqman qPCR were performed using the Taqman Advance miRNA assays for the following miRNAs (hsa-miR-1; has-miR-2, hsa-miR-3; hsa-miR-4, hsa-miR-5, hsa-miR-6; hsa-miR-7; hsa-miR-8; hsa-miR-9, has-miR-10). hsa-miR-10a-5p was used as internal control and Total RNA kidney (Thermosfisher) was used as positive control. Ct values of triplicates were averaged and used for &Dgr;Ct calculations. Principal component analysis (PCA), Receiver Operating Characteristic (ROC) analysis and Mann-Whitney test were done using GraphPrims v5 and SPSS v15. P<0.05 were set as significant. Results A total of 8 control samples and 14 pathological samples (3 IFTA, 6 ACR and 5 CIT) were processed by next generation sequencing. A total of 1500 miRNAs were identified, then after normalization and principal component analysis 12 miRNAs were selected to be tested in a new cohort. A new control group(N=8) and patients (IFTA: n=9; ACR: n=9; CIT: n=6) were selected as mentioned before. Then, uEVs and total RNA were isolated as described above. We found that eight miRNAs had different expression patterns in the urine of patients with any kind of graft pathology compared to controls. Conclusion miRNA profiling in urine has potential as a novel method for detecting pathology in kidney transplant.

Molecular profile of urine extracellular vesicles from normo-functional kidneys reveal minimal differences between living and deceased donors

BackgroundKidney transplantation (KTx) is the best therapeutic approach for chronic kidney diseases leading to irreversible kidney failure. Considering the origin of the graft, several studies have reported differences between living (LD) and deceased donors (DD) in graft and patient survival. These differences seem to be related to multiple factors including, donor age and time of cold ischemia among others. Many of transplanted organs come from old-aged DDs, in which pre-transplant biopsy is recommended. However, kidney biopsy has several limitations, and there is a need to develop alternatives to assess the status of a kidney before transplantation. As the analysis of urinary extracellular vesicles (uEVs) rendered promising results as non-invasive biomarkers of kidney-related pathologies, this pilot study aimed to investigate whether profiling uEVs of LDs and DDs may be of help to assess the quality of the kidney before nephrectomy.MethodsuEVs from 5 living donors and 7 deceased donors were isolated by size-exclusion chromatography, and their protein and miRNA content were analysed by liquid chromatography followed by mass spectrometry and next generation sequencing, respectively. Then, hierarchical clustering and venn diagrams were done with Perseus software and InteractiVenn tool. Specific EVs data bases were also used for Gene Ontology analysis.ResultsNext generation sequencing revealed that uEVs from DDs contained less miRNAs than LDs, but most of the DD-expressed miRNAs were shared with LDs (96%). Only miR-326 (targeting the apoptotic-related Bcl2) was found significantly over-represented in LD. Focusing on the protein content, we detected a low intra-group correlation in both types of donors. Despite these differences, hierarchical clustering of either miRNA or protein data could not identify a differential profile between LDs and DDs. Of note, 90% of transplanted patients had a functional graft after a year from KTx.ConclusionsIn this pilot study we found that, in normo-functional grafts, minor differences in uEVs profile could not discriminate between LDs and DDs.