Dextrin conjugation to colistin inhibits its toxicity, cellular uptake and acute kidney injury in vivo.

The acute kidney injury (AKI) and dose-limiting nephrotoxicity, which occurs in 20-60% of patients following systemic administration of colistin, represents a challenge in the effective treatment of multi-drug resistant Gram-negative infections. To reduce clinical toxicity of colistin and improve targeting to infected/inflamed tissues, we previously developed dextrin-colistin conjugates, whereby colistin is designed to be released by amylase-triggered degradation of dextrin in infected and inflamed tissues, after passive targeting by the enhanced permeability and retention effect. Whilst it was evident in vitro that polymer conjugation can reduce toxicity and prolong plasma half-life, without significant reduction in antimicrobial activity of colistin, it was unclear how dextrin conjugation would alter cellular uptake and localisation of colistin in renal tubular cells in vivo. We discovered that dextrin conjugation effectively reduced colistin's toxicity towards human kidney proximal tubular epithelial cells (HK-2) in vitro, which was mirrored by significantly less cellular uptake of Oregon Green (OG)-labelled dextrin-colistin conjugate, when compared to colistin. Using live-cell confocal imaging, we revealed localisation of both, free and dextrin-bound colistin in endolysosome compartments of HK-2 and NRK-52E cells. Using a murine AKI model, we demonstrated dextrin-colistin conjugation dramatically diminishes both proximal tubular injury and renal accumulation of colistin. These findings reveal new insight into the mechanism by which dextrin conjugation can overcome colistin's renal toxicity and show the potential of polymer conjugation to improve the side effect profile of nephrotoxic drugs.

miR-141 mediates recovery from acute kidney injury.

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 < 0.05) and decreases in miR-192 (p < 0.001) and miR-204 (p < 0.05). Expression of miR-141 increased in renal proximal tubular epithelial cells (PTECs) under oxidative stress in vitro and unilateral ischaemic reperfusion injury in vivo. 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.

Detection of urinary microRNA biomarkers using diazo sulfonamide-modified screen printed carbon electrodes.

This paper describes a straightforward electrochemical method for rapid and robust urinary microRNA (miRNA) quantification using disposable biosensors that can discriminate between urine from diabetic kidney disease (DKD) patients and control subjects. Aberrant miRNA expression has been observed in several major human disorders, and we have identified a urinary miRNA signature for DKD. MiRNAs therefore have considerable promise as disease biomarkers, and techniques to quantify these transcripts from clinical samples have significant clinical and commercial potential. Current RT-qPCR-based methods require technical expertise, and more straightforward methods such as electrochemical detection offer attractive alternatives. We describe a method to detect urinary miRNAs using diazo sulfonamide-modified screen printed carbon electrode-based biosensors that is amenable to parallel analysis. These sensors showed a linear response to buffered miR-21, with a 17 fM limit of detection, and successfully discriminated between urine samples (n = 6) from DKD patients and unaffected control subjects (n = 6) by differential miR-192 detection. Our technique for quantitative miRNA detection in liquid biopsies has potential for development as a platform for non-invasive high-throughput screening and/or to complement existing diagnostic procedures in disorders such as DKD.

Assessment of Urinary MicroRNAs by Quantitative Polymerase Chain Reaction in Diabetic Nephropathy Patients.

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.

Inhibition of Kirsten-Ras reduces fibrosis and protects against renal dysfunction in a mouse model of chronic folic acid nephropathy.

Chronic Kidney Disease is a growing problem across the world and can lead to end-stage kidney disease and cardiovascular disease. Fibrosis is the underlying mechanism that leads to organ dysfunction, but as yet we have no therapeutics that can influence this process. Ras monomeric GTPases are master regulators that direct many of the cytokines known to drive fibrosis to downstream effector cascades. We have previously shown that K-Ras is a key isoform that drives fibrosis in the kidney. Here we demonstrate that K-Ras expression and activation are increased in rodent models of CKD. By knocking down expression of K-Ras using antisense oligonucleotides in a mouse model of chronic folic acid nephropathy we can reduce fibrosis by 50% and prevent the loss of renal function over 3 months. In addition, we have demonstrated in vitro and in vivo that reduction of K-Ras expression is associated with a reduction in Jag1 expression; we hypothesise this is the mechanism by which targeting K-Ras has therapeutic benefit. In conclusion, targeting K-Ras expression with antisense oligonucleotides in a mouse model of CKD prevents fibrosis and protects against renal dysfunction.

A urinary microRNA panel that is an early predictive biomarker of delayed graft function following kidney transplantation.

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 < 0.05 for each). Receiver operating characteristic analysis showed Area Under the Curve 0.94 for this panel. To establish whether this early signal was sustained, miR-21 was measured daily for 5 days post-transplant, and was consistently elevated in those developing Delayed Graft Function (n = 165 samples from 33 patients, p < 0.05). The biomarker panel was then evaluated in an independent cohort, sampled at varying times in the first week post-transplantation in a separate transplant center. When considered individually, all miRs in the panel showed a trend to increase or a significant increase in those developing delayed Graft Function (miR-9: P = 0.068, mIR-10a: P = 0.397, miR-21: P = 0.003, miR-29a: P = 0.019, miR-221: P = 0.1, and miR-429: P = 0.013, n = 47) with Area Under the Curve 0.75 for the panel. In conclusion, combined measurement of six microRNAs had predictive value for delayed graft function following kidney transplantation.

Association of Elevated Urinary miR-126, miR-155, and miR-29b with Diabetic Kidney Disease.

Effective diabetic kidney disease (DKD) biomarkers remain elusive, and urinary miRNAs represent a potential source of novel noninvasive disease sentinels. We profiled 754 miRNAs in pooled urine samples from DKD patients (n = 20), detecting significantly increased miR-126, miR-155, and miR-29b compared with controls (n = 20). These results were confirmed in an independent cohort of 89 DKD patients, 62 diabetic patients without DKD, and 41 controls: miR-126 (2.8-fold increase; P < 0.0001), miR-155 (1.8-fold increase; P < 0.001), and miR-29b (4.6-fold increase; P = 0.024). Combined receiver operating characteristic curve analysis resulted in an area under the curve of 0.8. A relative quantification threshold equivalent to 80% sensitivity for each miRNA gave a positive signal for 48% of DKD patients compared with 3.6% of diabetic patients without DKD. Laser-capture microdissection of renal biopsy specimens, followed by quantitative RT-PCR, detected miR-155 in glomeruli and proximal and distal tubules, whereas miR-126 and miR-29b were most abundant in glomerular extracts. Subsequent experiments showed miR-126 and miR-29b enrichment in glomerular endothelial cells (GEnCs) compared with podocytes, proximal tubular epithelial cells, and fibroblasts. Significantly increased miR-126 and miR-29b were detected in GEnC conditioned medium in response to tumor necrosis factor-α and transforming growth factor-ß1, respectively. Our data reveal an altered urinary miRNA profile associated with DKD and link these variations to miRNA release from GEnCs.

Electrochemical detection of urinary microRNAs via sulfonamide-bound antisense hybridisation.

Altered serum and plasma microRNA (miRNA) expression profiles have been observed in numerous human diseases, with a number of studies describing circulating miRNA biomarkers for cancer diagnosis, prognosis and response to treatment, and recruitment to clinical trials for miRNA-based drug therapy already underway. Electrochemical detection of biomarkers in urine has several significant advantages over circulating biomarker analysis including safety, cost, speed and ease of conversion to the point of care environment. Consequently, much current research is underway to identify urinary miRNA biomarkers for a variety of pathologies including prostate and bladder malignancies, and renal disorders. We describe here a robust method capable of electrochemical detection of human urinary miRNAs at femtomolar concentrations using a complementary DNA-modified glassy carbon electrode. A miR-21-specific DNA hybridisation probe was immobilised onto a glassy carbon electrode modified by sulfonic acid deposition and subsequent chlorination. In our pilot system, the presence of synthetic mature miR-21 oligonucleotides increased resistance at the probe surface to electron transfer from the ferricyanide/ferrocyanide electrolyte. Response was linear for 10 nM-10 fM miR-21, with a limit of detection of 20 fM, and detection discriminated between miR-21, three point-mutated miR-21 sequences, and miR-16. We then demonstrated similar sensitivity and reproducibility of miR-21 detection in urine samples from 5 human control subjects. Our protocol provides a platform for future high-throughput screening of miRNA biomarkers in liquid biopsies.

Regulation of synthesis and roles of hyaluronan in peritoneal dialysis.

Hyaluronan (HA) is a ubiquitous extracellular matrix glycosaminoglycan composed of repeated disaccharide units of alternating D-glucuronic acid and D-N-acetylglucosamine residues linked via alternating ß-1,4 and ß-1,3 glycosidic bonds. HA is synthesized in humans by HA synthase (HAS) enzymes 1, 2, and 3, which are encoded by the corresponding HAS genes. Previous in vitro studies have shown characteristic changes in HAS expression and increased HA synthesis in response to wounding and proinflammatory cytokines in human peritoneal mesothelial cells. In addition, in vivo models and human peritoneal biopsy samples have provided evidence of changes in HA metabolism in the fibrosis that at present accompanies peritoneal dialysis treatment. This review discusses these published observations and how they might contribute to improvement in peritoneal dialysis.

Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein.

A pressing need for new chronic kidney disease (CKD) biomarkers persists. MicroRNAs (miRNAs) are emerging as a novel class of disease biomarkers in body fluids, but mechanisms conferring their stability in urine have not been fully elucidated. Here we investigated stabilization in human urine of ubiquitously expressed miR-16, and miR-192, which we have shown previously to be downregulated in renal fibrosis, by association with extracellular vesicles and with argonaute protein (AGO) 2. Endogenous urinary miR-16 was significantly more resistant to RNase-mediated degradation than exogenous, spiked-in, Caenorhabditis elegans cel-miR-39. We used our previously optimized high-resolution exosome isolation protocol with sucrose gradient ultracentrifugation to sub-fractionate the primary extracellular vesicle-rich urinary pellet. MiR-16 and miR-192 were enriched in exosomal sucrose gradient fractions, but were also detected in all other fractions. This suggested association of urinary miRNAs with other urinary extracellular vesicles and/or pellet components, complicating previous estimates of miRNA:exosome stoichiometry. Proteinase K digestion destabilized urinary miR-16 and we showed, for the first time, RNA-immunoprecipitation of urinary miR-16:AGO2 and miR-192:AGO2 complexes. Association with exosomes and AGO2 stabilized urinary miR-16 and miR-192, suggesting quantitative urinary miRNA analysis has the potential to identify novel, non-invasive CKD biomarkers.

Mutations in TJP2 cause progressive cholestatic liver disease.

Elucidating genetic causes of cholestasis has proved to be important in understanding the physiology and pathophysiology of the liver. Here we show that protein-truncating mutations in the tight junction protein 2 gene (TJP2) cause failure of protein localization and disruption of tight-junction structure, leading to severe cholestatic liver disease. These findings contrast with those in the embryonic-lethal knockout mouse, highlighting differences in redundancy in junctional complexes between organs and species.

Antisense knockdown of Kras inhibits fibrosis in a rat model of unilateral ureteric obstruction.

Tubulointerstitial fibrosis is the hallmark of chronic kidney disease and is characterized by an increase in the number and activity of interstitial fibroblasts and by excessive matrix deposition. Ras is an intracellular signaling molecule involved in cell proliferation and differentiation. It has recently been implicated in the pathogenesis of renal fibrosis. Of the three different isoforms of Ras (Kirsten, Harvey, and Neural), we previously demonstrated that the Kirsten isoform is key in the control of renal fibroblast proliferation in vitro. In this study, we used gene therapy in the form of antisense oligonucleotides (ASOs) specifically to silence Kras (alias Ki-ras) expression in a rat model of renal fibrosis caused by unilateral ureteric obstruction. We demonstrate that renal Kras expression increases by 70% in this model compared with sham-operated animals and that treatment with ASOs can reduce total renal Kras by >90% to levels well below basal. This silencing is associated with a dramatic inhibition of interstitial fibrosis, a fivefold reduction in α-smooth muscle actin expression, and a 2.4-fold reduction in collagen I deposition. This inhibition was observed despite histologic evidence of marked interstitial inflammation. These findings demonstrate that silencing Kras expression can markedly inhibit renal fibrosis. This strategy should be considered as a new potential therapeutic avenue.