Ioana Bancu

Urinary Extracellular Vesicles as Source of Biomarkers in Kidney Diseases

Most cells physiologically release vesicles as way of intercellular communication. The so-called Extracellular Vesicles (EVs) include exosomes, ectosomes, and apoptotic bodies, which basically differ in their composition and subcellular origin. Specifically, EVs found in urine reflect the state of the urinary system, from podocytes to renal-tubular cells, thus making them an excellent source of samples for the study of kidney physiology and pathology. Several groups have focused on defining biomarkers of kidney-related disorders, from graft rejection to metabolic syndromes. So far, the lack of a standard protocol for EVs isolation precludes the possibility of a proper comparison among the different biomarkers proposed in the literature, stressing the need for validation of these biomarkers not only in larger cohorts of patients but also considering the different methods for EVs isolation. In this review, we aim to gather the current knowledge about EVs-related biomarkers in kidney diseases, with a special emphasis in the methods used to date for EVs enrichment, and discussing the need for more specific protocols of EV isolation in clinical practice.

Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples

Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this invasive method is of limited repeatability and often describes an irreversible renal damage. Urine is an easily accessible fluid and urinary extracellular vesicles (EVs) may be ideal to describe new biomarkers associated with renal pathologies. Several methods to enrich EVs have been described. Most of them contain a mixture of proteins, lipoproteins and cell debris that may be masking relevant biomarkers. Here, we evaluated size-exclusion chromatography (SEC) as a suitable method to isolate urinary EVs. Following a conventional centrifugation to eliminate cell debris and apoptotic bodies, urine samples were concentrated using ultrafiltration and loaded on a SEC column. Collected fractions were analysed by protein content and flow cytometry to determine the presence of tetraspanin markers (CD63 and CD9). The highest tetraspanin content was routinely detected in fractions well before the bulk of proteins eluted. These tetraspanin-peak fractions were analysed by cryo-electron microscopy (cryo-EM) and nanoparticle tracking analysis revealing the presence of EVs. When analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, tetraspanin-peak fractions from urine concentrated samples contained multiple bands but the main urine proteins (such as Tamm–Horsfall protein) were absent. Furthermore, a preliminary proteomic study of these fractions revealed the presence of EV-related proteins, suggesting their enrichment in concentrated samples. In addition, RNA profiling also showed the presence of vesicular small RNA species. To summarize, our results demonstrated that concentrated urine followed by SEC is a suitable option to isolate EVs with low presence of soluble contaminants. This methodology could permit more accurate analyses of EV-related biomarkers when further characterized by -omics technologies compared with other approaches.

Long-Term Normal Renal Function after Drastic Weight Reduction in Patients with Obesity-Related Glomerulopathy

Aims: No long-term studies of renal function evolution in morbidly obese (MO) patients after weight loss are available. The aim of our work was to ascertain the long-term influence of drastic weight reduction on renal function in MO patients with obesity-related glomerular lesions. Methods: 92 MO patients with normal renal function and biopsy evidence of mild obesity-related glomerulopathy underwent bariatric surgery (BS) and subsequent drastic weight loss. A long-term prospective follow-up (mean duration: 76 ± 42 months) was carried out. Basal renal biopsies and basal and long-term metabolic and renal function studies were performed in all cases. Linear mixed models were applied. Results: Blood pressure dropped early after BS and remained stable thereafter. Creatinine clearance and BMI fell in the first 2 years, rose slightly after 5 years and then remained stable. Serum creatinine and albuminuria decreased throughout the follow-up period. Renal function and albuminuria evolution showed non-significant differences in relation to the number of glomerular lesions. Conclusions: Drastic weight loss in BS-treated MO patients with pre-surgical normal renal function and mild obesity-related glomerular lesions is associated with short- and long-term maintenance of normal renal function and improvement in both arterial hypertension and albuminuria.

Low Insulin-Like Growth Factor-1 Level in Obesity Nephropathy: A New Risk Factor?

Introduction IGF-1 (insulin-like growth factor-1) is a hormone involved in cell growth and other important processes. In the kidney, IGF-1 has a stimulating effect, increasing the blood flow and glomerular filtration rate. Although many experimental animal studies regarding the role of IGF-1 in the kidney have been conducted, few human studies are available in the literature. Obesity is a cause of renal failure, and several glomerular lesions associated with obesity have been described. However, no studies regarding the levels of IGF-1 in morbidly obese patients with renal injury associated with obesity have been conducted. Aim To determine the serum IGF-1 concentrations in morbidly obese patients with normal renal function but with different types of early obesity-related glomerular lesions and to evaluate the possible relationship between IGF-1 and the presence of renal lesions. Methods Eighty morbidly obese patients with renal biopsy, including 11 patients with no evidence of renal lesion, 17 patients with single glomerulomegaly, 21 patients with single podocyte hypertrophy, 10 patients with glomerulomegaly and podocyte hypertrophy, 5 patients with focal segmental hyalinosis, and 16 patients with increased mesangial matrix and/or mesangial proliferation, participated in this study. Biological parameters, including serum IGF-1 concentrations with the standard deviation score for age (SDS-IGF-1), were determined for all patients. Results Eighty patients (50 women and 30 men) with a mean BMI of 52.63 ± 8.71 and a mean age of 42.40 ± 9.45 years were included in this study. IGF-1, IGF-1 SDS and IGF-1BP3 levels according to the renal injury were compared (normal glomeruli: IGF-1 = 190.17 ± 72.46; glomerulomegaly: IGF-1 = 122.3 ± 50.05; podocyte hypertrophy: IGF-1 = 119.81 ± 60.34; focal segmental hyalinosis: IGF-1 170.98 ± 100.83, increased mesangial matrix and/or mesangial proliferation: IGF-1 117.73 ± 63.87). Statistically significant differences were observed between serum levels of IGF-1 and between the levels of SDS-IGF-1 by comparing the group without glomerular lesion with the group formed by patients with any type of glomerular injury. Logistic regression analysis was performed, with the dependent variable defined as the glomerular injury. In the multivariate analysis, only SDS-IGF-1 was associated with glomerular injury, and low levels of IGF-1 SDS were a risk factor for kidney injury. Conclusions Our study demonstrates that low IGF-1 serum levels are associated with renal lesions in morbidly obese patients without overt clinical renal manifestations.

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.

Frail Patient in Hemodialysis: A New Challenge in Nephrology—Incidence in Our Area, Barcelonès Nord and Maresme

Introduction Labeling a patient as “frail” may be useful in assessing the prognosis and therapeutic approach. Objective The aim of the study is to define a pattern of frailty among our dialysis population, to analyse the incidence and clinical evolution of these patients. Materials and Methods We analysed a total of 320 patients with stage V chronic kidney disease (CKD) who were on hemodialysis between September 2014 and September 2015. To define a patient as frail we used the Fried phenotype model, and we added a new criteria-dialysis session length longer than 12 hours/week. Results 5.6% of the 320 patients were frail. We found statistically significant differences regarding body mass index (BMI), hemoglobin (Hgb), and serum albumin, as well as the ability to perform the basic activities of daily living (p < 0.005), ability to ambulate (p = 0.01) and perform transfers (p < 0.005). We found statistically significant differences between the two groups in terms of hospital admissions (p = 0.005) and mortality (p < 0.005). Conclusion 5.6% of the study population were frail, with lower BMI, serum albumin and hemoglobin, lower capacity for basic activities of daily living, ambulation, and transference, as well as higher morbidity and mortality.