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Background After transplantation, cell-free deoxyribonucleic acid (DNA) derived from the donor organ (ddcfDNA) can be detected in the recipient’s circulation. We aimed to investigate the role of plasma ddcfDNA as biomarker for acute kidney rejection. Methods From 107 kidney transplant recipients, plasma samples were collected longitudinally after transplantation (Day 1 to 3 months) within a multicentre set-up. Cell-free DNA from the donor was quantified in plasma as a fraction of the total cell-free DNA by next generation sequencing using a targeted, multiplex polymerase chain reaction-based method for the analysis of single nucleotide polymorphisms. Results Increases of the ddcfDNA% above a threshold value of 0.88% were significantly associated with the occurrence of episodes of acute rejection (P = 0.017), acute tubular necrosis (P = 0.011) and acute pyelonephritis (P = 0.032). A receiver operating characteristic curve analysis revealed an equal area under the curve of the ddcfDNA% and serum creatinine of 0.64 for the diagnosis of acute rejection. Conclusions Although increases in plasma ddcfDNA% are associated with graft injury, plasma ddcfDNA does not outperform the diagnostic capacity of the serum creatinine in the diagnosis of acute rejection.

BackgroundAfter transplantation, cell-free DNA derived from the donor organ (ddcfDNA) can be detected in the recipient’s circulation. We aimed to quantify ddcfDNA levels in plasma of kidney transplant recipients thereby investigating the kinetics of this biomarker after transplantation and determining biological variables that influence ddcfDNA kinetics in stable and non-stable patients.Materials and methodsFrom 107 kidney transplant recipients, plasma samples were collected longitudinally after transplantation (day 1–3 months) within a multicenter set-up. Cell-free DNA from the donor was quantified in plasma as a fraction of the total cell-free DNA by next generation sequencing using a targeted, multiplex PCR-based method for the analysis of single nucleotide polymorphisms. A subgroup of stable renal transplant recipients was identified to determine a ddcfDNA threshold value.ResultsIn stable transplant recipients, plasma ddcfDNA% decreased to a mean (SD) ddcfDNA% of 0.46% (± 0.21%) which was reached 9.85 (± 5.6) days after transplantation. A ddcfDNA threshold value of 0.88% (mean + 2SD) was determined in kidney transplant recipients. Recipients that did not reach this threshold ddcfDNA value within 10 days after transplantation showed a higher ddcfDNA% on the first day after transplantation and demonstrated a higher individual baseline ddcfDNA%.ConclusionIn conclusion, plasma ddcfDNA fractions decreased exponentially within 10 days after transplantation to a ddcfDNA threshold value of 0.88% or less. To investigate the role of ddcfDNA for rejection monitoring of the graft, future research is needed to determine causes of ddcfDNA% increases above this threshold value.

Kidney transplantation is considered the favored treatment for patients suffering from end-stage renal disease, since successful transplantation is associated with longer survival and improved quality of life compared to dialysis. Alloreactive immune responses against the donor kidney may lead to acute rejection of the transplant. The current diagnosis of renal allograft rejection mainly relies on clinical monitoring, including serum creatinine, proteinuria, and confirmation by histopathologic assessment in the kidney transplant biopsy. These parameters have their limitations. Identification and validation of biomarkers, which correlate with or predict the presence of acute rejection, and which could improve therapeutic decision making, are priorities for the transplantation community. There is a need for alternative, less invasive but sensitive markers to diagnose acute graft rejection. Here, we provide an overview of the current status on research of biomarkers of acute kidney transplant rejection in blood and urine. We specifically discuss relatively novel research strategies in biomarker research, including transcriptomics and proteomics, and elaborate on donor-derived cell-free DNA as a potential biomarker.

MicroRNAs are epigenetic regulators of gene expression at the posttranscriptional level. They are involved in intercellular communication and crosstalk between different organs. As key regulators of homeostasis, their dysregulation underlies several morbidities including kidney disease. Moreover, their remarkable stability in plasma and urine makes them attractive biomarkers. Beyond biomarker studies, clinical microRNA research in nephrology in recent decades has focused on the discovery of specific microRNA signatures and the identification of novel targets for therapy and/or disease prevention. However, much of this research has produced equivocal results and there is a need for standardization and confirmation in prospective trials. This review aims to provide an overview of general concepts and available clinical evidence in both the pathophysiology and biomarker fields for the role of microRNA in AKI and kidney transplantation. LDL Ago HDL MV Mature miRNA miRISC miRNA duplex Pre-miRNA Transcription MV MV MV E AB Interstitium Active release Passive release Capillary Ago Dicer XPO-5 Pri-miRNA Mirtron RNA pol II Drosha-DGCR8 Figure 1. | MicroRNA biogenesis and function.microRNA coding regions in the human genome are found either intergenic or in the introns of annotated genes. microRNA synthesis starts in the nucleus where most of the miRs are transcribed by RNA polymerase II into primary miR transcripts (pri-miR) of several kilobases that contain local stem-loop structures. The first step of miR maturation is cleavage at the stem of the hairpin structure by a microprocessor complex consisting of Drosha (an RNase III protein) together with its cofactor DiGeorge Syndrome Critical Region 8 (DGCR8), which releases a small hairpin structure of 70 nucleotides that is termed a precursor miR (pre-miR). After nuclear processing, pre-miRs are exported to the cytoplasm by exportin 5 (XPO-5), where they are cleaved near the terminal loop by another RNase enzyme called Dicer, thereby releasing an approximately 22 nucleotide miR duplex. This duplex is loaded onto an AGO protein to generate the microRNA-induced silencing complex (miRISC). One strand (guide strand) remains in the AGO protein as a biologically active miR whereas the other stand (passenger strand, known as miR*) is degraded. The mature miR as part of the effector RISC binds to the 39UTR region of the mRNA and mediates mRNA degradation, destabilization, or translational inhibition. Apart from this canonical pathway, there is an alternative "mirtron" pathway, independent from Drosha and DGCR8. Mirtrons are miRs that originate from spliced-out introns and are created when small RNAs bind to the termini of small intronic hairpins. Pre-microRNA hairpins with 39 overhangs are so formed and can mature into 22 nucleotides structures, which look and function as normal miRs. microRNAs exert their repressive function intracellularly, but are also released into the extracellular compartment, with this initiating theirroleasimportantintercellular communicators as theyare ta...

Purpose Real-time continuous glucose monitoring (RT-CGM) provides information on glycaemic variability (GV), time in range (TIR) and guidance to avoid hypoglycemia, thereby complimenting HbA1c for diabetes management. We investigated whether GV and TIR were independently associated with chronic and acute diabetes complications. Methods Between September 2014 and January 2017 515 subjects with type 1 diabetes using sensor-augmented pump therapy were followed for 24 months. The link between baseline HbA1c and CGM-derived glucometrics (TIR [70-180 mg/dL], coefficient of variation [CV] and standard deviation [SD]) obtained from the first 2 weeks of RT-CGM use and the presence of complications was investigated. Complications were defined as: composite microvascular complications (presence of neuropathy, retinopathy or nephropathy), macrovascular complications, and hospitalization for hypoglycemia and/or ketoacidosis. Results Individuals with microvascular complications were older (P<0.001), had a longer diabetes duration (P<0.001), a higher HbA1c (7.8±0.9 vs 7.5±0.9%, P<0.001) and spent less time in range (60.4±12.2 vs 63.9±13.8%, P=0.022) compared to those without microvascular complication. Diabetes duration (OR=1.12 [1.09-1.15],P<0.001) and TIR (OR=0.97 [0.95-0.99], P=0.005) were independent risk factors for composite microvascular complications, while SD and CV were not. Age (OR=1.08 [1.03-1.14],P=0.003) and HbA1c (OR=1.80 [1.02-3.14], P=0.044) were risk factors for macrovascular complications. TIR (OR=0.97 [0.95-0.99], P=0.021) was the only independent risk factor for hospitalizations for hypoglycaemia or ketoacidosis. Conclusions Lower TIR was associated with the presence of composite microvascular complications and with hospitalization for hypoglycemia or ketoacidosis. TIR, SD and CV were not associated with macrovascular complications.

Exercise training is an effective way to improve exercise capacity in chronic kidney disease (CKD), but the underlying mechanisms are only partly understood. In healthy subjects (HS), microRNA (miRNA or miR) are dynamically regulated following exercise and have, therefore, been suggested as regulators of cardiovascular adaptation to exercise. However, these effects were not studied in CKD before. The effect of acute exercise (i.e., an acute exercise bout) was assessed in 32 patients with CKD and 12 age- and sex-matched HS ( study 1). miRNA expression in response to chronic exercise (i.e., a 3-mo exercise training program) was evaluated in 40 CKD patients ( study 2). In a subgroup of study 2, the acute-exercise induced effect was evaluated at baseline and at follow-up. Plasma levels of a preselected panel miRNA, involved in exercise adaptation processes such as angiogenesis (miR-126, miR-210), inflammation (miR-21, miR-146a), hypoxia/ischemia (miR-21, miR-210), and progenitor cells (miR-150), were quantified by RT-PCR. Additionally, seven miRNA involved in similar biological processes were quantified in the subgroup of study 2. Baseline, studied miRNA were comparable in CKD and HS. Following acute exercise, miR-150 levels increased in both CKD (fold change 2.12 ± 0.39, P = 0.002; and HS: fold change 2.41 ± 0.48 P = 0.018, P for interaction > 0.05). miR-146a acutely decreased in CKD (fold change 0.92 ± 0.13, P = 0.024), whereas it remained unchanged in HS. Levels of miR-21, miR-126, and miR-210 remained unaltered. Chronic exercise did not elicit a significant change in the studied miRNA levels. However, an acute exercise-induced decrease in miR-210 was observed in CKD patients, only after training (fold change 0.76 ± 0.15). The differential expression in circulating miRNA in response to acute and chronic exercise may point toward a physiological role in cardiovascular adaptation to exercise, also in CKD.

Renal magnesium (Mg(2+)) and sodium (Na(+)) loss are well-known side effects of cyclosporine (CsA) treatment in humans, but the underlying mechanisms still remain unclear. Recently, it was shown that epidermal growth factor (EGF) stimulates Mg(2+) reabsorption in the distal convoluted tubule (DCT) via TRPM6 (Thébault S, Alexander RT, Tiel Groenestege WM, Hoenderop JG, Bindels RJ. J Am Soc Nephrol 20: 78-85, 2009). In the DCT, the final adjustment of renal sodium excretion is regulated by the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), which is activated by the renin-angiotensin-aldosterone system (RAAS). The aim of this study was to gain more insight into the molecular mechanisms of CsA-induced hypomagnesemia and hyponatremia. Therefore, the renal expression of TRPM6, TRPM7, EGF, EGF receptor, claudin-16, claudin-19, and the NCC, and the effect of the RAAS on NCC expression, were analyzed in vivo in a rat model of CsA nephrotoxicity. Also, the effect of EGF administration on these parameters was studied. CsA significantly decreased the renal expression of TRPM6, TRPM7, NCC, and EGF, but not that of claudin-16 and claudin-19. Serum aldosterone was significantly lower in CsA-treated rats. In control rats treated with EGF, an increased renal expression of TRPM6 together with a decreased fractional excretion of Mg(2+) (FE Mg(2+)) was demonstrated. EGF did not show this beneficial effect on TRPM6 and FE Mg(2+) in CsA-treated rats. These data suggest that CsA treatment affects Mg(2+) homeostasis via the downregulation of TRPM6 in the DCT. Furthermore, CsA downregulates the NCC in the DCT, associated with an inactivation of the RAAS, resulting in renal sodium loss.