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

Chronic kidney disease is a public health issue, causing an important mortality rate and high economic impact [1]. Currently, different replacement therapies for end-stage renal disease include haemodialysis, peritoneal dialysis and kidney transplantation (KTx). Of these, only KTx improves the quality of life of the patients, and is clearly better compared to dialysis regarding patient survival [2‐4].

KTx may be performed using organs from living (LD) or deceased donors (DD). Although the number of LDs has increased in the late years [5], the majority of transplanted organs come from aged DDs in a state of neurological death. Some of the most important parameters affecting patient’s survival after transplantation are related to age (of both donor and recipient), previous pathology of the recipient and the extent of the ischemia-reperfusion injury [4, 6‐8]. It is widely accepted, and strongly supported, that transplantation from LDs offers better long-term outcomes than deceased organ transplants [9‐11]; because of DDs are usually older, have more comorbidities and longer cold ischemia time than LD [10‐12]. Moreover, in order to maintain the organs in optimal conditions, hemodynamic stability, adequate oxygenation, the correction of hypothermia, diabetes insipidus and electrolyte disturbances should be corrected by drug administration [13]. For these purposes, DDs might receive dopamine, noradrenalin and vasopressin among other drugs. In addition, specific antibiotic treatment might be administered if infection is suspected [14]. All these maintaining interventions may modify the quality of the organ.

To date, the quality of the organ before nephrectomy has only been estimated by sonographic images and kidney biopsy. In fact, kidney biopsy is recommended in expanded criteria donors, including those aged over 60 years, or showing hypertension, diabetes and in non-beating heart donors [15]. Nevertheless, kidney biopsy requires the invasive sampling of the organ and thus might affect also its quality. Therefore, alternative non-invasive techniques are needed to determine the organ status and predict the KTx outcome.

In this context, Extracellular Vesicles (EVs) have emerged as a source of non-invasive biomarkers for several diseases [16, 17]. In particular, urine EVs (uEVs) are viewed as a subcellular image of the glomerular and tubular systems, and changes in their composition may reflect ongoing events occurring in the renal system [18‐20]. In this sense, a growing number of studies have proposed several EV-related biomarkers for kidney dysfunction, graft rejection [18, 21‐23], or chronic kidney disease [21, 24, 25]. Also, a recently published study analysed EVs from graft-preservation fluid to predict delayed graft function [26]. However, no reports to date have investigated the uEVs profiles from LD and DD organs to distinguish their quality status prior to transplantation. We aim to profile uEV form living and deceased donors to define their RNA and protein content before KTx.

In this pilot study, we explored the miRNA and protein content of uEVs from LD and DD kidney donors. Using size-exclusion chromatography (SEC) as a minimally altering isolation technique to obtain uEVs [27], we report the great similarity in the molecular profile of uEVs from LDs and DDs. Importantly, this correlated with the kidney function of the transplanted patients, which showed normal renal function 1 year after transplantation. In addition, we have identified several previously non-described uEVs miRNAs.

The study protocols were approved by the Clinical Research Ethics Committee of the Germans Trias i Pujol University Hospital and conformed to the principles outlined in the Declaration of Helsinki. No statistical power calculation was conducted prior to the study, size sample was estimated based on previous publications [28‐30].

In this pilot study, we aimed to characterise the miRNA and protein profiles of SEC-isolated uEVs from living and deceased kidney donors to profile the organ status before Kidney transplantation (KTx). Several factors account for the outcome of KTx, including those related to the characteristics of the donor [2‐4, 11]. Although one of the most important factors directly affecting KTx outcome is the time of cold ischemia, which is usually longer in DD organs, an additional factor to be considered is the status of the organ before the extraction. This could be affected by several factors such as age, donor general condition, or cause of death in DDs. In these aspects, the cohort of patients included in this pilot study shared the same characteristics of larger reported cohorts in terms of mean age and cold ischemia time in DD.

Acceptance of a given organ for transplantation is currently based on clinical and histopathological parameters. The latter are based on a kidney biopsy prior to transplantation, performed only under some circumstances -such as donors aged > 60 years old, or suffering from cardiovascular disorders-, with the aim to verify the quality of the kidney. Yet, as biopsy sampling variability and differences in criteria of analysis may lead to different assessment of the quality of the organ [49, 50], gathering additional information (such that found in uEVs) may be of help defining clearer criteria for organ acceptance.

Given that organ transplant donor characteristics are evolving to older donors (most of them with chronic pathologies) pre-transplant biopsy in these expanded criteria donors is highly recommended to evaluate the state of the organ. As kidney biopsies have limitations, there is a need to improve the analysis of the kidney status prior to transplantation. In this context, the content of uEVs that come from the excretory system may be of interest. Several studies have already shown that urinary EVs may contain specific signatures which may potentially serve as biomarkers of disease [20]. Similarly, normal kidneys also produce EVs, and their specific profile may be indicative of the physiological status of each particular organ. Profiling some specific markers found in urinary EVs may be of clear interest in this particular scenario as a source of non-invasive diagnostic and prognostic biomarkers on the kidney status before nephrectomy.

Most of uEV-related parameters, such as size, concentration and the presence of classical EV markers -including tetraspanins CD63 and CD9- were equally detected in LD and DD samples. Moreover we did not detect in our DD-uEVs preparations any contamination of apoptotic bodies-such as the presence of histones-, which could be expected after the inflammatory state produce by brain death [13, 51]. In this sense, the absence of plasma EVs markers (such as CD5L or moesin), supports that urinary derived EVs are mainly analysed.

Focusing on the study of the RNA content of SEC-isolated uEVs, and in line with previous studies based on ultracentrifuged samples [52], miRNAs and, to a lesser extent, tRNA and rRNA, were among the most abundant RNA species in uEVs. Given the prominent role of miRNAs in cell communication and regulation, we focused on their profile in LD and DD. Although most miRNAs found in DDs were also present in LDs, up to 24% of miRNAs (mainly related to cell communication and signal transduction) were detected only in LDs. The miRNA target prediction showed that those genes related with cell adhesion, vesicle mediated transport and cytoskeleton organization were overrepresented in LD compared to DD, while genes related to regulation of metabolism were underrepresented. These data may be suggestive of an alteration in intracellular trafficking and metabolism in DD. Yet, due to the complexity of miRNA regulation processes and the multiples targets described for a given specific miRNA, further studies are needed to reveal the functional implication of each of them in the specific nephrologic context.

In fact, only one miRNA (miR-326) was found to be over-represented in LD group. miR-326 was described previously in EVs derived from cancer cell lines [53], plasma [54] and endothelial cells [55], and it has been described to target bcl–xL, a member of the bcl-2 family, inducing apoptosis in human platelets [56]. It has also been reported that down-regulation of miR-326 may be involved in chemoresistance in lung cancer [57], poor prognosis and metastatic gastric cancer [58, 59] and osteosarcoma [60]. But the presence of this miRNA was not found before in uEVs and its role in the urinary track remains unknown.

Additionally, we further detected eleven miRNAs not described previously in EVs databases which are predicted to target genes related with the urinary tract. Once again, SEC is confirmed as confident method for isolating uEVs isolated and can be used for EV-related RNA studies and points out to their relevance in normal kidney function.

Focusing on the proteomic studies, and as seen in the miRNA data, a first observation was the identification of higher number of proteins in LDs compared to DDs. In addition, intra-group analyses demonstrated a higher level of variability of protein expression among DDs, which could be related to intrinsic or extrinsic factors such as the donor-conditioning regime. Despite the minimal differences detected between LD and DD in the proteomic and miRNA analysis, in this study all transplanted organs were functional 1 year after KTx, indicating that these minimal changes seem not to be of major relevance for organ function.

Currently, the majority of transplanted kidneys are obtained from aged DDs in a state of neurological death. The use of drugs and medical intervention to maintain these donors in optimal conditions may alter the organ, as suggested by Nemati et al. [61]. Our results show that, before nephrectomy, uEVs derived from DDs showing a 1 year follow-up normal functioning kidneys, contained minimal alterations in their miRNA composition compared to uEVs from LDs. On the other hand, although the DD protein content was highly heterogeneous; such differences did not correlate with the short-term (1 year) graft survival. Additional studies, in a larger cohort of donors and recipients, are needed to investigate whether the information contained in uEVs from subclinical altered kidneys could anticipate any detrimental effect on the graft after transplantation, as recently suggested using preservation fluids [26]. Despite there are some limitations in our pilot study, such as a reduced number of samples and a high variability found among donors, we have successfully profiled uEVs from kidney donors. These results although preliminary, open the possibility to analyse donor uEVs in search of potential biomarkers of kidney quality before nephrectomy.

Based on uEVs miRNA and proteomic data, no major differences could be detected between LDs and DDs, suggesting that the physiological status of the well-functioning organs before nephrectomy was similar in both groups.