Search

Background: For patients on peritoneal dialysis (PD), the deleterious effects of high concentrations of dialysate glucose on the peritoneal membrane are well-documented. Systemic effects of peritoneally absorbed glucose are more poorly defined. Using continuous glucose monitoring (CGM), we aimed to describe 24-h glycaemic profiles of PD patients without diabetes and compare with non-dialysis controls with stage 5 chronic kidney disease (CKD-5). Methods: In this cross-sectional, case-control study, 15 patients on PD (9 automated PD (APD) and 6 continuous ambulatory PD (CAPD)) and 16 CKD-5 controls underwent 72 h of CGM and metabolic profiling. CGM was used to derive average glucose concentrations and within-participant standard deviation (SD) of glucose. Data were analysed for the whole 72-h monitoring period and as daytime (09.00 to 21.00) and night-time (21.00 to 09.00). Results: Average glucose concentrations and within-participant SD of glucose for the whole monitoring period were not different between the three groups ( p ≥ 0.5). Daytime average glucose concentrations were also similar across the three groups ( p = 0.729). APD was associated with a significantly higher nocturnal glucose than CAPD (5.25 mmol/L ± 0.65 vs. 4.28 ± 0.5, p = 0.026). A significant drop in nocturnal glucose compared with daytime average seen in both CAPD patients and controls was absent in APD patients. Conclusions: Systematically different glycaemic patterns were observed in non-diabetic APD and CAPD patients, including an absence of physiological nocturnal glucose dipping in patients on APD. Comprehensive CGM data sets highlight subtleties not appreciated by traditional metabolic biomarkers; this has implications when choosing the most appropriate outcome measures in future research addressing the metabolic impact of PD.

Background Proximal tubular cells (PTCs) are the most abundant cell type in the kidney. PTCs are central to normal kidney function and to regeneration versus organ fibrosis following injury. This study used single-nucleus RNA sequencing (snRNAseq) to describe the phenotype of PTCs in renal fibrosis.\ud\udMethods Kidneys were harvested from naïve mice and from mice with renal fibrosis induced by chronic aristolochic acid administration. Nuclei were isolated using Nuclei EZ Lysis buffer. Libraries were prepared on the 10× platform, and snRNAseq was completed using the Illumina NextSeq 550 System. Genome mapping was carried out with high-performance computing.\ud\udResults A total of 23,885 nuclei were analyzed. PTCs were found in five abundant clusters, mapping to S1, S1–S2, S2, S2-cortical S3, and medullary S3 segments. Additional cell clusters (“new PTC clusters”) were at low abundance in normal kidney and in increased number in kidneys undergoing regeneration/fibrosis following injury. These clusters exhibited clear molecular phenotypes, permitting labeling as proliferating, New-PT1, New-PT2, and (present only following injury) New-PT3. Each cluster exhibited a unique gene expression signature, including multiple genes previously associated with renal injury response and fibrosis progression. Comprehensive pathway analyses revealed metabolic reprogramming, enrichment of cellular communication and cell motility, and various immune activations in new PTC clusters. In ligand-receptor analysis, new PTC clusters promoted fibrotic signaling to fibroblasts and inflammatory activation to macrophages.\ud\udConclusions These data identify unrecognized PTC phenotype heterogeneity and reveal novel PTCs associated with kidney fibrosis.

Infectious peritonitis is a common complication in patients undergoing chronic peritoneal dialysis (PD), limiting the duration of PD as a modality for renal replacement therapy and increasing patient morbidity and mortality. Antimicrobial peptides (AMPs) serve critical roles in mucosal defense, but their expression and activity during peritonitis are poorly understood. We hypothesized that AMPs belonging to the Ribonuclease (RNase) A Superfamily are present in peritoneal fluid and increase during peritonitis in patients undergoing chronic PD. In the absence of peritonitis, we detected RNase 3, RNase 6, and RNase 7 in cell-free supernatants and viable cells obtained from peritoneal fluid of chronic PD patients. The cellular sources of these RNases were eosinophils (RNase 3), macrophages (RNase 6), and mesothelial cells (RNase 7). During peritonitis, RNase 3 increased 55-fold and RNase 7 levels increased 3-fold on average, whereas RNase 6 levels were unchanged. The areas under the receiver-operating characteristic curves for RNase 3 and RNase 7 were 0.99 (95% confidence interval (CI): 0.96–1.0) and 0.79 (95% CI: 0.64–0.93), respectively, indicating their potential as biomarkers of peritonitis. Discrete omental reservoirs of these RNases were evident in patients with end stage kidney disease prior to PD initiation, and omental RNase 3 reactive cells increased in patients undergoing PD with a history of peritonitis. We propose that constitutive and inducible pools of antimicrobial RNases form a network to shield the peritoneal cavity from microbial invasion in patients undergoing chronic PD.

Acute kidney injury (AKI) is a global clinical problem characterised by a sudden decline in renal function and mortality as high as 60%. Current AKI biomarkers have limited ability to classify disease progression and identify underlying pathological mechanisms. Here we hypothesised that alterations in urinary microRNA profiles could predict AKI recovery/nonrecovery after 90 days, and that injury-specific changes would signify microRNA mediators of AKI pathology. Comparison of urinary microRNA profiles from AKI patients with controls detected significant injury-specific increases in miR-21, miR-126 and miR-141 (p . Forced miR-141 expression in the presence of H2O2 increased PTEC death and decreased cell viability. Of nine messenger RNA targets with two or more miR-141 3’-untranslated region binding sites, we confirmed protein tyrosine phosphatase receptor type G (PTPRG) as a direct miR-141 target in PTECs. PTPRG-specific siRNA knockdown under oxidative stress increased PTEC death and decreased cell viability. In conclusion, we detected significant alterations in five urinary microRNAs following AKI, and identified proximal tubular cell PTPRG as a putative novel therapeutic target.

Ischemic preconditioning (IPC) is effective in limiting subsequent ischemic acute kidney injury in experimental models. MicroRNAs are an important class of post-transcriptional regulator and show promise as biomarkers of kidney injury. We evaluated the time- and dose-dependence of benefit from IPC in a rat model of functional (bilateral) ischemia–reperfusion injury (IRI). We found optimal protection from subsequent injury following short, repetitive sequences of preconditioning insult. We subsequently used hybridization array and microRNA sequencing to characterize microRNA signatures of protective IPC and of IRI. These approaches identified a profile of microRNA changes consequent on IRI, that were limited by prior IPC. To localize these signals within the kidney, we used laser capture microdissection and RT-qPCR to measure microRNA abundance in nephron segments, pinpointing microRNA changes principally to glomeruli and proximal tubules. Our data describe a unique microRNA signature for IRI in the rat kidney. Pulsatile IPC reduces kidney damage following IRI and diminishes this microRNA signal. We have also identified candidate microRNAs that may act as biomarkers of injury and therapeutic targets in this context.

International audience; Retinoic acid (RA) activates RA receptors (RAR), resulting in RA response element (RARE)-dependent gene expression in renal collecting duct (CD). Emerging evidence supports a protective role for this activity in acute kidney injury (AKI) and chronic kidney disease (CKD). Herein, we examined this activity in RARE-LacZ transgenic mice and by RARE-Luciferase reporter assays in CD cells, and investigated how this activity responds to neurotransmitters and mediators of kidney injury. In RARE-LacZ mice, Adriamycin-induced heavy albuminuria was associated with reduced RA/RAR activity in CD cells. In cultured CD cells, RA/RAR activity was repressed by acetylcholine, albumin, aldosterone, angiotensin II, high glucose, cisplatin and lipopolysaccharide, but was induced by aristolochic acid I, calcitonin gene-related peptide, endothelin-1, gentamicin, norepinephrine and vasopressin. Compared with age-matched normal human CD cells, CD-derived renal cystic epithelial cells from patients with autosomal recessive polycystic kidney disease (ARPKD) had significantly lower RA/ RAR activity. Synthetic RAR agonist RA-568 was more potent than RA in rescuing RA/RAR activity repressed by albumin, high glucose, angiotensin II, aldosterone, cisplatin and lipopolysaccharide. Hence, RA/RAR in CD cells is a convergence point of regulation by neurotransmitters and mediators of kidney injury, and may be a novel therapeutic target. Acute kidney injury (AKI) is an abrupt renal insult followed by sudden decline of kidney functions 1,2 ; chronic kidney disease (CKD) is a long-term condition where the kidneys do not work effectively, resulting in chronic loss of renal functions 3,4. AKI occurs in 10-15% hospitalised patients and approximately 50% patients in intensive care, and is associated with high mortality 2 ; affecting 8-16% of the world's population 3,4 , CKD was the 12th most common cause of mortality and caused 4.6% of deaths globally in 2017 5. If this rising prevalence of CKD continues, it is predicted that CKD will become a top-5 cause of mortality worldwide by 2040 6. AKI and CKD are not distinct disease entities but rather are closely interconnected syndromes 7. Despite some shared aetiologies, risk factors and mechanisms, the pathophysiology of AKI and CKD can be quite different. Although AKI may be caused by systemic, vascular, glomerular and tubular diseases, the core pathophysiology of AKI is often predominated by tubulointerstitial injury 2. On the other hand, although a wide range of aetiologies may all cause CKD, the latter is most often a glomerular disease 4. However, regardless of the primary aetiologies of CKD, tubulointerstitial injury plays a crucial role in CKD progression to end-stage kidney disease 8. Thus, it is critical to investigate the mechanism of tubulointerstitial injury, which is as yet poorly understood. What we open

Neutrophil infiltration is a hallmark of peritoneal inflammation, but mechanisms regulating neutrophil recruitment in patients with peritoneal dialysis (PD)-related peritonitis are not fully defined. We examined 104 samples of PD effluent collected during acute peritonitis for correspondence between a broad range of soluble parameters and neutrophil counts. We observed an association between peritoneal IL-17 and neutrophil levels. This relationship was evident in effluent samples with low but not high IFN-γ levels, suggesting a differential effect of IFN-γ concentration on neutrophil infiltration. Surprisingly, there was no association of neutrophil numbers with the level of CXCL1, a key IL-17-induced neutrophil chemoattractant. We investigated therefore the production of CXCL1 by human peritoneal mesothelial cells (HPMCs) under in vitro conditions mimicking clinical peritonitis. Stimulation of HPMCs with IL-17 increased CXCL1 production through induction of transcription factor SP1 and activation of the SP1-binding region of the CXCL1 promoter. These effects were amplified by TNFα. In contrast, IFN-γ dose-dependently suppressed IL-17-induced SP1 activation and CXCL1 production through a transcriptional mechanism involving STAT1. The SP1-mediated induction of CXCL1 was also observed in HPMCs exposed to PD effluent collected during peritonitis and containing IL-17 and TNFα, but not IFN-γ. Supplementation of the effluent with IFN-γ led to a dose-dependent activation of STAT1 and a resultant inhibition of SP1-induced CXCL1 expression. Transmesothelial migration of neutrophils in vitro increased upon stimulation of HPMCs with IL-17 and was reduced by IFN-γ. In addition, HPMCs were capable of binding CXCL1 at their apical cell surface. These observations indicate that changes in relative peritoneal concentrations of IL-17 and IFN-γ can differently engage SP1-STAT1, impacting on mesothelial cell transcription of CXCL1, whose release and binding to HPMC surface may determine optimal neutrophil recruitment and retention during peritonitis. © 2020 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Background and Aims Microvascular impairment is an early step in cardiovascular disease (CVD), the major cause of morbidity and mortality in patients with CKD. Changes in skin microvascular reactivity have been shown to reflect more widespread changes in the systemic and coronary microcirculation. Microvascular function is attenuated by advancing age and conditions that commonly coexist with CKD such as diabetes and hypertension. We investigated whether skin microvascular reactivity is impaired in peritoneal dialysis (PD) patients compared with healthy controls and whether this impairment is independent of comorbidity. Method Forearm skin vasculature was examined in 27 healthy controls, 27 patients on PD and 27 controls matched to the PD patients for age, gender, diabetes and previous CV events. Microvascular function was assessed using laser Doppler flowmetry in combination with post-occlusive reactive hyperaemia (a test of generalised microvascular function) and iontophoretic application of acetylcholine (ACh) and sodium nitroprusside (SNP) to investigate endothelium dependant and non-endothelium dependant vasodilation respectively. Results Peak post-occlusive flow was lower in the PD patients than in both the healthy controls and the co-morbidity matched group; 90.45 AU (60.9-128.35) in healthy controls, 73AU (58.8-134.4) in matched controls and 56.95AU (45.1-89.8) in PD patients (p=0.0239 healthy controls versus patients, p=0.0373 matched controls versus patients). SNP-mediated vasodilatation was statistically lower in the PD group compared with healthy controls (p= 0.016), ACh response was lower but did not reach statistical significance (p= 0.076). ACh and SNP-mediated vasodilatation trended towards being lower in PD patients than matched controls but did not reach statistical significance. Conclusion The PD patients were characterised by a generalised dysfunction of the skin microcirculation compared with healthy controls and controls matched for factors known to affect the microcirculation. This appears to be the result of impaired endothelium dependant and independent vasodilatory mechanisms.

Understanding why certain patients with IgA nephropathy progress to kidney failure while others maintain normal kidney function remains a major unanswered question. To help answer this, we performed miRNome profiling by next generation sequencing of kidney biopsies in order to identify microRNAs specifically associated with the risk of IgA nephropathy progression. Following sequencing and validation in independent cohorts, four microRNAs (-150-5p, -155-5p, -146b-5p, -135a-5p) were found to be differentially expressed in IgA nephropathy progressors compared to non-progressors, and patients with thin membrane nephropathy, lupus nephritis and membranous nephropathy, and correlated with estimated glomerular filtration rate, proteinuria, and the Oxford MEST-C scores (five histological features that are independent predictors of clinical outcome). Each individual microRNA increased the discrimination score of the International IgAN Prediction Tool, although due to the small number of samples the results did not reach statistical significance. miR-150-5p exhibited the largest amplitude of expression between cohorts and displayed the best discrimination between IgA nephropathy progressors and non-progressors by receiver operating curve analysis (AUC: 0.8). However, expression was similarly upregulated in kidneys with established fibrosis and low estimated glomerular filtration rates at the time of biopsy. Consistent with a more generic role in kidney fibrosis, in situ hybridization revealed that miR-150-5p was found in lymphoid infiltrates, and areas of proliferation and fibrosis consistent with the known drivers of progression. Thus, miR-150-5p may be a potential functional mediator of kidney fibrosis that may add value in predicting risk of progression in IgA nephropathy and other kidney diseases.

Urinary microRNAs show promise as noninvasive biomarkers in renal disease. Here, we describe a detailed protocol for the column-based extraction and quantification of miRNAs by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) from urine samples.

Infection remains a major cause of morbidity, mortality and technique failure in patients with end stage kidney failure who receive peritoneal dialysis (PD). Recent research suggests that the early inflammatory response at the site of infection carries diagnostically relevant information, suggesting that organ and pathogen-specific “immune fingerprints” may guide targeted treatment decisions and allow patient stratification and risk prediction at the point of care. Here, we recorded microRNA profiles in the PD effluent of patients presenting with symptoms of acute peritonitis and show that elevated peritoneal miR-223 and reduced miR-31 levels were useful predictors of bacterial infection. Cell culture experiments indicated that miR-223 was predominantly produced by infiltrating immune cells (neutrophils, monocytes), while miR-31 was mainly derived from the local tissue (mesothelial cells, fibroblasts). miR-223 was found to be functionally stabilised in PD effluent from peritonitis patients, with a proportion likely to be incorporated into neutrophil-derived exosomes. Our study demonstrates that microRNAs are useful biomarkers of bacterial infection in PD-related peritonitis and have the potential to contribute to disease-specific immune fingerprints. Exosome-encapsulated microRNAs may have a functional role in intercellular communication between immune cells responding to the infection and the local tissue, to help clear the infection, resolve the inflammation and restore homeostasis.

Peritoneal dialysis (PD) remains limited by dialysis failure due to peritoneal membrane fibrosis driven by inflammation caused by infections or sterile cellular stress. Given the fundamental role of Toll-like receptors (TLRs) and complement in inflammation, we assessed the potential of peritoneal TLR2, TLR4 and C5a receptors, C5aR and C5L2, as therapeutic targets in PD-associated fibrosis. We detected TLR2–, TLR4–, and C5aR–mediated proinflammatory and fibrotic responses to bacteria that were consistent with the expression of these receptors in peritoneal macrophages (TLR2/4, C5aR) and mesothelial cells (TLR2, C5aR). Experiments in knockout mice revealed a major role for TLR2, a lesser role for TLR4, a supplementary role for C5aR, and no apparent activity of C5L2 in infection–induced peritoneal fibrosis. Similarly, antibody blockade of TLR2, TLR4, or C5aR differentially inhibited bacteria–induced profibrotic and inflammatory mediator production by peritoneal leukocytes isolated from the peritoneal dialysis effluent (PDE) of noninfected uremic patients. Additionally, antibodies against TLR2, TLR4, or the coreceptor CD14 reduced the profibrotic responses of uremic leukocytes to endogenous components present in the PDE of noninfected patients. Enhancing TLR2-mediated inflammation increased fibrosis in vivo. Furthermore, soluble TLR2 (sTLR2), a negative modulator of TLRs that we detected in PDE, inhibited PDE–induced, TLR2– or TLR4–mediated profibrotic responses. Notably, sTLR2 treatment markedly reduced Gram–positive and –negative bacteria–induced fibrosis in vivo, inhibiting proinflammatory and fibrotic genes without affecting infection clearance. These findings reveal the influence of peritoneal TLR2 and TLR4 on PD-associated fibrosis and describe a therapeutic strategy against fibrosis.

Long-term peritoneal dialysis (PD) often results in the development of peritoneal fibrosis. In many other fibrosing diseases, monocytes enter the fibrotic lesion and differentiate into fibroblast-like cells called fibrocytes. We find that peritoneal tissue from short-term PD patients contains few fibrocytes, while fibrocytes are readily observed in the peritoneal membrane of long-term PD patients. The PD fluid Dianeal (Baxter Healthcare Corporation, Deerfield, IL, USA) contains dextrose, a number of electrolytes including sodium chloride, and sodium lactate. We find that PD fluid potentiates human fibrocyte differentiation in vitro and implicates sodium lactate in this potentiation. The plasma protein serum amyloid P (SAP) inhibits fibrocyte differentiation. Peritoneal dialysis fluid and sodium chloride decrease the ability of human SAP to inhibit human fibrocyte differentiation in vitro. Together, these results suggest that PD fluid contributes to the development of peritoneal fibrosis by potentiating fibrocyte differentiation.

Chronic kidney disease (CKD) is a health problem reaching epidemic proportions. There is no cure for CKD, and patients may progress to end-stage renal disease (ESRD). Peritoneal dialysis (PD) is a current replacement therapy option for ESRD patients until renal transplantation can be achieved. One important problem in long-term PD patients is peritoneal membrane failure. The mechanisms involved in peritoneal damage include activation of the inflammatory and immune responses, associated with submesothelial immune infiltrates, angiogenesis, loss of the mesothelial layer due to cell death and mesothelial to mesenchymal transition, and collagen accumulation in the submesothelial compact zone. These processes lead to fibrosis and loss of peritoneal membrane function. Peritoneal inflammation and membrane failure are strongly associated with additional problems in PD patients, mainly with a very high risk of cardiovascular disease. Among the inflammatory mediators involved in peritoneal damage, cytokine IL-17A has recently been proposed as a potential therapeutic target for chronic inflammatory diseases, including CKD. Although IL-17A is the hallmark cytokine of Th17 immune cells, many other cells can also produce or secrete IL-17A. In the peritoneum of PD patients, IL-17A-secreting cells comprise Th17 cells, gamma delta T cells, mast cells, and neutrophils. Experimental studies demonstrated that IL-17A blockade ameliorated peritoneal damage caused by exposure to PD fluids. This article provides a comprehensive review of recent advances on the role of IL-17A in peritoneal membrane injury during PD and other PD-associated complications. This research was funded by grants from the Instituto de Salud Carlos III (ISCIII) and Fondos FEDER European Union (PI17/00119 to M.R.-O.; PI17/01495, DTS17/00203, and DTS19/00093 to J.E.; PI16/02057 and PI19/00815 to A.O.; PI 15/00120 to R.S; PI18/0610 to J.M.V.; and Red de Investigacion Renal (REDINREN): RD16/0009 to M.R.-O., A.O., R.S., and J.M.V.); by Comunidad de Madrid ("NOVELREN" B2017/BMD-3751 to M.R.-O. and B2017/BMD-3686 CIFRA2-CM to A.O.); by the "Juan de la Cierva Formacion" training program of the Ministerio de Economia, Industria y Competitividad (MINECO) supporting the salary of S.R.-M. (FJCI-2016-29050); by "Convocatoria Dinamizacion Europa Investigacion 2019" MINECO (EIN2019-103294 to M.R.-O. and S.R.-M.); by Sociedad Espanola de Nefrologia (S.E.N. to M.R.-O.); by ERA-PerMed-JTC2018 (KIDNEY ATTACK AC18/00064 and PERSTIGAN AC18/00071) and DTS18/00032 toA.O; by IMPROVE-PD project ("Identification and Management of Patients at Risk-Outcome and Vascular Events in Peritoneal Dialysis") with funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 812699 to M.R.O., M.L.-C., and D.F.; and by the Spanish Ministry of Science and Innovation/Fondo Europeo de Desarrollo Regional (MICINN/FEDER) (PID2019-110132RB-I00) to M.L.-C.

Peritoneal membrane failure due to fibrosis limits the use of peritoneal dialysis (PD). Peritoneal fibrosis may potentially be induced by sterile inflammation caused by ongoing cellular stress due to prolonged exposure to PD solutions (PDS). Effective therapies to prevent this process remain to be developed. Toll-like receptors (TLRs) mediate sterile inflammation by recognizing damage-associated molecular patterns (DAMPs) released by cellular stress. We evaluated the involvement of TLRs and DAMPs in PDS-induced fibrosis models and the therapeutic potential of TLR-DAMP targeting for preventing fibrosis. A range of PDS elicited pro-inflammatory and fibrotic responses from PD patient peritoneal leukocytes, mesothelial cells and mouse peritoneal leukocytes. TLR2/4 blockade of human peritoneal cells or TLR2/4 knockouts inhibited these effects. PDS did not induce rapid ERK phosphorylation or IκB-α degradation, suggesting that they do not contain components capable of direct TLR activation. However, PDS increased the release of Hsp70 and hyaluronan, both TLR2/4 DAMP ligands, by human and mouse peritoneal cells, and their blockade decreased PDS-driven inflammation. Soluble TLR2, a TLR inhibitor, reduced PDS-induced pro-inflammatory and fibrotic cytokine release ex vivo. Daily catheter infusion of PDS in mice caused peritoneal fibrosis, but co-administration of soluble TLR2 prevented fibrosis, suppressed pro-fibrotic gene expression and pro-inflammatory cytokine production, reduced leukocyte/neutrophil recruitment, recovered Treg cell levels and increased the Treg:Th17 ratio. Thus, TLR2/4, Hsp70 and hyaluronan showed major roles in PDS-induced peritoneal inflammation and fibrosis. The study demonstrates the therapeutic potential of a TLR-DAMP targeting strategy to prevent PDS-induced fibrosis.

Predicting immediate and subsequent graft function is important in clinical decision-making around kidney transplantation, but is difficult using available approaches. Here we have evaluated urinary microRNAs as biomarkers in this context. Profiling of 377 microRNAs in the first urine passed post-transplantation identified 6 microRNAs, confirmed to be upregulated by RT-qPCR in an expanded cohort (miR-9, -10a, -21, -29a, -221, and -429, n = 33, P

Hypothermic machine perfusion is effective in improving outcome following kidney transplantation. Molecular analyses of hypothermic machine perfusate (HMP) have the potential to identify biomarkers of organ viability prior to transplantation, offering significant advantages to the transplant surgeon, and leading to a potential increase in the organ donor pool. MicroRNAs are emerging as important biomarkers in the context of kidney injury and transplantation. Recent data demonstrate increased microRNA-21 (miR-21) expression in the kidney following acute kidney injury. This study investigated the potential of miR-21 detected in HMP to act as a sentinel for early kidney transplant outcomes. MiR-21 was found to be readily detectable in HMP by RT-qPCR. Eleven ECD kidneys were maintained on a hypothermic machine perfusion system for a median 627 (range 117-1027) minutes, and evaluation of flow and resistance characteristics suggested stability on the machine from 60 min post-perfusion. MiR-21 quantification at 60 min post-perfusion correlated with eGFR at 6 and 12 months post-transplantation. These data suggest that miR-21 expression in HMP may be predictive of early outcomes following kidney transplantation. In the era of ECD kidneys, a reliable measure of organ quality is urgently needed, and this study suggests miR-21 may be such a marker.

Peritoneal dialysis (PD) therapy substantially requires biomarkers as tools to identify patients who are at the highest risk for PD-related complications and to guide personalized interventions that may improve clinical outcome in the individual patient. In this consensus article, members of the European Training and Research in Peritoneal Dialysis Network (EuTRiPD) review the current status of biomarker research in PD and suggest a selection of biomarkers that can be relevant to the care of PD patients and that are directly accessible in PD effluents. Currently used biomarkers such as interleukin-6, interleukin-8, ex vivo–stimulated interleukin-6 release, cancer antigen-125, and advanced oxidation protein products that were collected through a Delphi procedure were first triaged for inclusion as surrogate endpoints in a clinical trial. Next, novel biomarkers were selected as promising candidates for proof-of-concept studies and were differentiated into inflammation signatures (including interleukin-17, M1/M2 macrophages, and regulatory T cell/T helper 17), mesothelial-to-mesenchymal transition signatures (including microRNA-21 and microRNA-31), and signatures for senescence and inadequate cellular stress responses. Finally, the need for defining pathogen-specific immune fingerprints and phenotype-associated molecular signatures utilizing effluents from the clinical cohorts of PD patients and “omics” technologies and bioinformatics-biostatistics in future joint-research efforts was expressed. Biomarker research in PD offers the potential to develop valuable tools for improving patient management. However, for all biomarkers discussed in this consensus article, the association of biological rationales with relevant clinical outcomes remains to be rigorously validated in adequately powered, prospective, independent clinical studies.

Purpose of review: This review summarizes recent data supporting the concept that urinary microRNAs are a useful new class of biomarker. They may improve capacity to stratify patients with chronic kidney disease according to risk of progression, and may also inform about response to therapy.\ud \ud Recent findings: MicroRNAs are present, stable and readily quantifiable in tissues and body fluids, including urine, and have widespread importance as regulators in the kidney. Urinary microRNAs are typically released from the nephron or downstream structures, and their abundance may reflect altered microRNA expression in the kidney, or release into the lumen by the cells comprising the different regions of the nephron. As a consequence, abundance of specific microRNAs in the urine may change in various pathological states. Large-scale studies are now needed, to test the capacity of specific microRNAs to inform about risk and response to therapy.\ud \ud Summary: Urinary microRNAs appear useful sentinels for pathological processes occurring in the kidney and may enable a ‘personalized medicine’ approach to the management and stratification of renal disease

Peritoneal dialysis (PD) is a life-saving form of renal replacement therapy for those with end-stage kidney disease. Mesothelial cells (MCs) line the peritoneal cavity and help define peritoneal response to treatment-associated injury, a major reason for treatment failure. miRNAs are important regulators, but their roles in peritoneal fibrosis are largely unknown. In this study, miR-21 was one of the most abundant miRNAs in primary MCs, and was up-regulated by the profibrotic cytokine transforming growth factor-β1 and in PD effluent-derived MCs exhibiting mesenchymal phenotypic change. Increased miR-21 was found in peritoneal membrane biopsy specimens from PD patients compared to healthy controls (PD biocompatible, 5.86×, P = 0.0001; PD conventional, 7.09×, P n = 11 per group). In PD effluent from a cohort of 230 patients, miR-21 was higher in those receiving the therapy long-term compared to new starters ( n = 230, miR-21 3.26×, P = 0.001) and associated with icodextrin use ( R = 0.52; 95% CI, 0.20–0.84), peritonitis count ( R = 0.16; 95% CI, 0.03–0.29), and dialysate cytokines. miR-21 down-regulated programmed cell death 4 and programmed cell death 4 protein was decreased in peritoneal membrane biopsy specimens from PD patients compared to healthy controls. New miR-21 targets were identified that may be important during PD fibrogenesis. These data identify miR-21 as an important effector of fibrosis in the peritoneal membrane, and a promising biomarker in the dialysis effluent for membrane change in patients receiving PD.

Mesothelial cell (MC) senescence contributes to malignancy and tissue fibrosis. The\ud role of telomere erosion in MC senescence remains controversial, with evidence for\ud both telomere-dependent and telomere-independent mechanisms reported. Single\ud telomere length analysis revealed considerable telomere length heterogeneity in\ud freshly isolated human peritoneal MCs, reflecting a heterogeneous proliferative history\ud and providing high-resolution evidence for telomere-dependent senescence. By\ud contrast the attenuated replicative lifespan, lack of telomere erosion and induction of\ud p16 expression in in vitro-aged cells was consistent with stress-induced senescence.\ud Given the potential pathophysiological impact of senescence in mesothelial tissues,\ud high-resolution MC telomere length analysis may provide clinically useful information.

Recent estimates suggest that 1 in 12 of the global population suffers from diabetes mellitus. Approximately 40 % of those affected will go on to develop diabetes-related chronic kidney disease or diabetic nephropathy (DN). DN is a major cause of disability and premature death. Existing tests for prognostic purposes are limited and can be invasive, and interventions to delay progression are challenging. MicroRNAs (miRNAs) are a recently described class of molecular regulators found ubiquitously in human tissues and bodily fluids, where they are highly stable. Alterations in miRNA expression profiles have been observed in numerous diseases. Blood and tissue miRNAs are already established cancer biomarkers, and cardiovascular, metabolic and immune disease miRNA biomarkers are under development. Urinary miRNAs represent a potential novel source of non-invasive biomarkers for kidney diseases, including DN. In addition, recent data suggest that miRNAs may have therapeutic applications. Here, we review the utility of miRNAs as biomarkers for the early detection and progression of DN, assess emerging data on miRNAs implicated in DN pathology and discuss how the data from both fields may contribute to the development of novel therapeutic agents.

Peritoneal dialysis is a widely used mode of renal replacement therapy in which preservation of the structural and functional integrity of the peritoneal membrane is critical for continued success. Progressive scarring, or fibrosis, in the peritoneal membrane is now well described in peritoneal dialysis patients, but its extent is variable. While some patients survive for long periods on peritoneal dialysis, others suffer from ‘fibrosis-related’ membrane failure, or rarely, more severe complications such as encapsulating peritoneal sclerosis (EPS). The reasons for these variations in responses are unlikely to be explained by variations in the therapy, which has remained largely unchanged over the past 20 years, and are suggestive of a genetic component to the susceptibility of individuals to different outcomes. In this issue of NDT, Margetts et al. [1] describe how they used genetically different mouse strains to examine the variability in responses to a defined, constant pro-fibrotic stimulus in a model of peritoneal fibrosis. The data highlight a possible genetic linkage to susceptibility to fibrosis that adds significant insights into our understanding of the mechanisms driving peritoneal damage. The key to continued success of peritoneal dialysis as a therapy remains in preservation of the performance of the peritoneal membrane as a dialysing organ. In health, the visceral and parietal peritoneum and its phospholipid-rich secretions and anti-friction surfaces facilitate bowel motility within the abdominal cavity. The outer surface of the whole comprises a single layer of mesothelial cells, specialized to provide a low friction and non-adhesive surface. The mesothelium that lines the peritoneal membrane sits on the sub-mesothelial compact zone, comprising a collagen-rich extracellular matrix in which larger blood vessels and capillaries are sited. Peritoneal dialysis is associated with a spectrum of alterations in the morphology of the peritoneal membrane which includes alterations in the mesothelial cell morphology, thickening of the sub-mesothelial compact zone and progressive vascular damage (vasculopathy) [2]. Cross-sectional studies have linked these changes to long-term glucose exposure and episodic infection [2].These alterations to the peritoneal membrane associate with alterations in peritoneal solute transport, loss of ultrafiltration and eventual technique failure. More recent data suggest that the changes in the mesothelium play a key role in driving the changes in peritoneal structure and function. Data primarily from animal models and corroborated with the limited clinical studies suggest that the acquisition of a fibroblast-like phenotype by mesothelial cells, so-called trans-differentiation or epithelial-mesenchymal transition, is key in driving changes in extracellular matrix turnover and fibrogenesis (reviewed in [3]). As the genesis of peritoneal fibrosis is insidious without overt clinical symptoms, except in cases of EPS, there are at present no tests that can predict its onset, nor are there any therapeutic approaches other than a switch of renal replacement therapy modality from peritoneal dialysis to haemodialysis when membrane function is seriously compromised. In the case of EPS, surgery can be used to relieve intestinal obstruction, but while outcomes have improved from this intervention the risk of mortality from the condition remains significant. The clinical consequences of peritoneal fibrosis are thus clear, but the processes that initiate and drive it in peritoneal

MicroRNAs are short noncoding RNA regulators that repress synthesis of their targets post-transcriptionally. On average, each microRNA is estimated to regulate several hundred protein-coding genes, and about 60% of proteins are thought to be regulated by microRNAs in total. A subset of these genes, including the key profibrotic cytokine transforming growth factor beta-1 (TGF-β1), exhibits particularly strong levels of post-transcriptional control of protein synthesis, involving microRNAs and other mechanisms. Changes in microRNA expression pattern are linked to profound effects on cell phenotype, and microRNAs have an emerging role in diverse physiological and pathological processes. In this review, we provide an overview of microRNA biology with a focus on their emerging role in diseases typified by organ fibrosis.

Kidney biopsy is the gold-standard diagnostic test for intrinsic renal disease, but requires hospital admission and carries a 3% risk of major complications. Current non-invasive prognostic indicators such as urine protein quantification have limited predictive value. Better diagnostic and prognostic tests for chronic kidney disease patients are a major focus for industry and academia, with efforts to date directed largely at urinary proteomic approaches. microRNAs constitute a recently identified class of endogenous short non-coding single-stranded RNA oligonucleotides that regulate gene expression post-transcriptionally. Quantification of urinary microRNAs offers an alternative approach to the identification of chronic kidney disease biomarkers.

The kidneys play key roles in the maintenance of homeostasis, including fluid balance, blood filtration, erythropoiesis and hormone production. Disease-driven perturbation of renal function therefore has profound pathological effects, and chronic kidney disease is a leading cause of morbidity and mortality worldwide. Successive annual increases in global chronic kidney disease patient numbers in part reflect upward trends for predisposing factors, including diabetes, obesity, hypertension, cardiovascular disease and population age. Each kidney typically possesses more than one million functional units called nephrons, and each nephron is divided into several discrete domains with distinct cellular and functional characteristics. A number of recent analyses have suggested that signaling between these nephron regions may be mediated by microRNAs. For this to be the case, several conditions must be fulfilled: (i) microRNAs must be released by upstream cells into the ultrafiltrate; (ii) these microRNAs must be packaged protectively to reach downstream cells intact; (iii) these packaged microRNAs must be taken up by downstream recipient cells without functional inhibition. This review will examine the evidence for each of these hypotheses and discuss the possibility that this signaling process might mediate pathological effects.

Renal replacement therapy (RRT) is necessary for the survival of patients with end-stage renal disease (ESRD) both prior to kidney transplantation or in patients where kidney transplantation is not available. Peritoneal dialysis (PD) and hemodialysis (HD) are lifesaving RRTs for more than 2 million patients with ESRD worldwide [1]. As the incidence of chronic renal disease has doubled over the past decade, the number of patients with ESRD is expected to increase by 5–8% annually [2]. Although long-term morbidity and mortality are comparable between PD and HD, there is an early patient survival advantage for PD and a better quality of life [3]. Moreover PD is cost-effective as compared to hospital-based HD [4]. Nevertheless only one out of 10 patients is treated with PD, which suggests a general underutilization [5]. PD is a simple therapy in which PD fluid is exchanged several times a day; the major limitations are peritoneal membrane damage on the long-term and infection [6]. PD could be largely enhanced if one can identify diagnostic and therapeutic tools to improve PD outcome that promote function of the peritoneal membrane and prevent infectious complications [7]. European Training and Research in Peritoneal Dialysis (EuTRiPD) is an EU funded training programme, in which exactly these goals are targeted [8]. In this special issue of BMRI, we present a number of articles prepared in part (but also by other contributors) with a support from the EuTRiPD initiative. These are both original research papers and review papers that (i) analyze basic aspects of peritoneal cell biology using in vitro methods, (ii) mimic clinical situations in relevant PD models in rodents, and (iii) describe the outcomes of clinical studies using new biomarkers or new interventions. In the following papers, firstly studies apply in vitro models to investigate the senescence-associated proteome in mesothelial cells, the effect of differently sterilized PD fluids on mesothelial stress responses, and the contribution of mesothelial-to-mesenchymal transition to peritoneal fibroblast expansion. Secondly with respect to papers in rodent PD models a new mouse uremic model is presented, the effect of rapamycin in a mouse model is shown, and moreover clear indications are described in protective effect of paricalcitol in a PD model and another PD-fibrosis-like model. Thirdly clinical material was used in studies showing the importance of, respectively, HA, microRNA, osmolarity of fluid, and tenascin-C levels reflecting peritoneal patient status. Finally underutilization of PD is discussed as originating from less education and referral pattern and moreover it is discussed that new biomarkers in PD could contribute significantly to making choices for the future as shown by EuTRiPD. The editors feel that all these papers give a good overview of the state of art in addressing all aspects of PD. Robert H. J. Beelen Donald J. Fraser Peter Rutherford Janusz Witowski

miRNAs are small, endogenous, post-transcriptional regulators of gene expression. AKI (acute kidney injury) of various aetiologies, including trauma, sepsis and IRI (ischaemia/reperfusion injury) in the context of kidney transplantation, or drug toxicity, has a high morbidity and mortality rate and presents a significant burden to health services worldwide. AKI primarily affects the renal cortex, in particular PTCs (proximal tubular epithelial cells). Current research demonstrates causality between G2/M cell cycle arrest of PTCs and AKI. Recent findings from our laboratory and others presented in this review implicate miRNA regulation of the cell cycle in the pathology of AKI.

In this issue, Loureiro, and Cabrera et al. study the regulation of transforming growth factor beta-1 (TGF-β) signalling by bone morphogenetic protein-7 (BMP-7) in the biology of peritoneal dialysis-related peritoneal fibrosis. Peritoneal dialysis (PD) is a highly effective and convenient mode of renal replacement therapy, the success of which depends on the structural and functional integrity of the peritoneal membrane. Progressive alterations in peritoneal membrane transport characteristics commonly cause technique failure in PD, and are associated with fibroproliferative changes in the peritoneal membrane, including vasculopathy, accumulation of submesothelial extracellular matrix, and denudation and altered appearance of mesothelial cells [1]. The fundamental causes of these changes are likely to be long-term exposure to bioincompatible dialysate, together with recurrent episodes of bacterial peritonitis. Fibroproliferative diseases are largely driven by fibroblasts, spindle-shaped, motile, contractile cells that respond to pro-fibrotic stimuli by synthesizing and organizing extracellular matrix. Fibroblasts may originate from proliferation of local tissue fibroblasts, from circulating bone marrow-derived precursors (fibrocytes) and from resident epithelial cells by epithelial-to-mesenchymal transition (EMT) [2]. During EMT, epithelial cells lose intercellular adhesiveness and apical–basal polarity, and acquire a motile and contractile phenotype [3]. High-glucose concentrations typical of peritoneal dialysate induce such phenotypic change in mesothelial cells in vitro [4]. Additionally, the previous work of Cabrera et al. demonstrated progressive loss of epithelial morphology in mesothelial cells

Aristolochic acid nephropathy is characterized by rapidly progressive tubulointerstitial nephritis culminating in end-stage renal failure and urothelial malignancy. Profibrotic effects of aristolochic acid are linked to growth arrest of proximal tubular epithelial cells; however, the underlying mechanisms are largely undetermined. miRNAs are small, endogenous, post-transcriptional regulators of gene expression implicated in numerous physiological and pathological processes. In the present study, we characterized the mechanism of aristolochic acid-induced cell cycle arrest and its regulation by miRNAs. Incubation with aristolochic acid led to profound G2/M arrest in proximal tubular epithelial cells via p53-mediated inactivation of the maturation-promoting complex, CDK1/cyclin-B1. Analysis of miRNA expression identified up-regulation of miRNAs, including miR-192, miR-194, miR-450a, and miR-542-3p. The stable overexpression of miR-192 recapitulated G2/M arrest via repression of the E3 ubiquitin ligase, murine double-minute 2, a negative regulator of p53. p53-induced transcription of p21(cip1) and growth arrest and DNA damage 45 and resulted in the inactivation and dissociation of the maturation-promoting complex. These data demonstrate a core role for miR-192 in mediating proximal tubular epithelial cell G2/M arrest after toxic injury by aristolochic acid. Because numerous studies have linked such growth arrest to fibrosis after proximal tubular epithelial cell injury, this mechanism may have widespread relevance to recovery/nonrecovery after acute kidney injury.

Diverse aetiologies result in significant deviation from homoeostasis in the kidney, leading to CKD (chronic kidney disease). CKD progresses to end-stage renal disease principally as a result of renal fibrosis, although the molecular mechanisms underlying this fibrotic process are still poorly understood. miRNAs (microRNAs) are a recently discovered family of endogenous short single-stranded RNAs that regulate global gene expression at the post-transcriptional level. The recent findings from our laboratory and others discussed in the present review outline pleiotropic roles for miR-192 in renal homoeostasis and in the fibrotic kidney. We describe miR-192-driven anti-and pro-fibrotic effects via the repression of ZEB1 and ZEB2 (zinc finger E-box-binding homeobox proteins 1 and 2), resulting in changes in extracellular matrix deposition and cell differentiation.

Bone morphogenetic protein-6 (BMP-6) suppresses inflammatory genes in renal proximal tubular cells and regulates iron metabolism by inducing hepcidin. In diabetic patients, an increase of myofibroblast progenitor cells (MFPCs), also known as fibrocytes, was found to be associated with decreased BMP-6 expression. We hypothesized that loss of endogenous BMP-6 would aggravate renal injury and fibrosis. Wild type (WT) and BMP-6 null mice underwent unilateral ureteral obstruction. In WT mice, ureteral obstruction down-regulated BMP-6. Obstructed kidneys of BMP-6 null mice showed more casts (1.5-fold), epithelial necrosis (1.4-fold), and brush border loss (1.3-fold). This was associated with more inflammation (1.8-fold more CD45(+) cells) and more pronounced overexpression of profibrotic genes for αSMA (2.0-fold), collagen I (6.8-fold), fibronectin (4.3-fold), CTGF (1.8-fold), and PAI-1 (3.8-fold), despite similar BMP-7 expression. Also, 1.3-fold more MFPCs were obtained from BMP-6 null than from WT mononuclear cell cultures, but in vivo only very few MFPCs were observed in obstructed kidneys, irrespective of BMP-6 genotype. The obstructed kidneys of BMP-6 null mice showed 2.2-fold more iron deposition, in association with 3.3-fold higher expression of the oxidative stress marker HO-1. Thus, ureteral obstruction leads to down-regulation of BMP-6 expression, and BMP-6 deficiency aggravates tubulointerstitial damage and fibrosis independent of BMP-7. This process appears to involve loss of both direct anti-inflammatory and antifibrotic action and indirect suppressive effects on renal iron deposition, oxidative stress, and MFPCs.

The role of microRNAs (miRs), which are endogenous RNA oligonucleotides that regulate gene expression, in diabetic nephropathy is unknown. Here, we performed expression profiling of cultured proximal tubular cells (PTCs) under high-glucose and control conditions. We identified expression of 103 of 328 microRNAs but did not observe glucose-induced changes in expression. Next, we performed miR expression profiling in pooled RNA from formalin-fixed, paraffin-embedded tissue from renal biopsies. We studied three groups of patients with established diabetic nephropathy and detected 103 of 365 miRs. Two miRs differed by more than two-fold between progressors and nonprogressors, and 12 miRs differed between late presenters and other biopsies. We noted the greatest change in miR-192 expression, which was significantly lower in late presenters. Furthermore, in individual biopsies, low expression of miR-192 correlated with tubulointerstitial fibrosis and low estimated GFR. In vitro, treatment of PTCs with TGF-β1 decreased miR-192 expression. Overexpression of miR-192 suppressed expression of the E-Box repressors ZEB1 and ZEB2, thereby opposing TGF-β–mediated downregulation of E-cadherin. In summary, loss of miR-192 expression associates with increased fibrosis and decreased estimated GFR in diabetic nephropathy in vivo, perhaps by enhancing TGF-β–mediated downregulation of E-cadherin in PTCs.