Robust Rat and Mouse Models of Bilateral Renal Ischemia Reperfusion Injury.

BACKGROUND/AIM: Acute and chronic kidney diseases are a major contributor to morbidity and mortality worldwide, with no specific treatments currently available for these. To enable understanding the pathophysiology of and testing novel treatments for acute and chronic kidney disease, a suitable in vivo model of kidney disease is essential. In this article, we describe two reliable rodent models (rats and mice) of efficacious kidney injury displaying acute to chronic kidney injury progression, which is also reversible through novel therapeutic strategies such as ischemic preconditioning (IPC). MATERIALS AND METHODS: We utilized adult male Lewis rats and adult male wildtype (C57BL/6) mice, performed a midline laparotomy, and induced warm ischemia to both kidneys by bilateral clamping of both renal vascular pedicles for a set time, to mimic the hypoxic etiology of disease commonly found in kidney injury. RESULTS: Bilateral ischemia reperfusion injury caused marked structural and functional kidney injury as exemplified by histology damage scores, serum creatinine levels, and kidney injury biomarker levels in both rodents. Furthermore, this effect displayed a dose-dependent response in the mouse model. CONCLUSION: These rodent models of bilateral kidney IRI are reliable, reproducible, and enable detailed mechanistic study of the underlying pathophysiology of both acute and chronic kidney disease. They have been carefully optimised for single operator use with a strong track record of training both surgically trained and surgically naïve operators.

Tuning the Fermi Level of Graphene by Two-Dimensional Metals for Raman Detection of Molecules.

Graphene-enhanced Raman scattering (GERS) offers great opportunities to achieve optical sensing with a high uniformity and superior molecular selectivity. The GERS mechanism relies on charge transfer between molecules and graphene, which is difficult to manipulate by varying the band alignment between graphene and the molecules. In this work, we synthesized a few atomic layers of metal termed two-dimensional (2D) metal to precisely and deterministically modify the graphene Fermi level. Using copper phthalocyanine (CuPc) as a representative molecule, we demonstrated that tuning the Fermi level can significantly improve the signal enhancement and molecular selectivity of GERS. Specifically, aligning the Fermi level of graphene closer to the highest occupied molecular orbital (HOMO) of CuPc results in a more pronounced Raman enhancement. Density functional theory (DFT) calculations of the charge density distribution reproduce the enhanced charge transfer between CuPc molecules and graphene with a modulated Fermi level. Extending our investigation to other molecules such as rhodamine 6G, rhodamine B, crystal violet, and F16CuPc, we showed that 2D metals enabled Fermi level tuning, thus improving GERS detection for molecules and contributing to an enhanced molecular selectivity. This underscores the potential of utilizing 2D metals for the precise control and optimization of GERS applications, which will benefit the development of highly sensitive, specific, and reliable sensors.

Identification and detection of microRNA kidney disease biomarkers in liquid biopsies.

PURPOSE OF REVIEW: MicroRNAs (miRNAs) are emerging rapidly as a novel class of biomarkers of major organ disorders, including kidney diseases. However, current PCR-based detection methods are not amenable to development for high-throughput, cost-effective miRNA biomarker quantification. RECENT FINDINGS: MiRNA biomarkers show significant promise for diagnosis and prognosis of kidney diseases, including diabetic kidney disease, acute kidney injury, IgA nephropathy and delayed graft function following kidney transplantation. A variety of novel methods to detect miRNAs in liquid biopsies including urine, plasma and serum are being developed. As miRNAs are functional transcripts that regulate the expression of many protein coding genes, differences in miRNA profiles in disease also offer clues to underlying disease mechanisms. SUMMARY: Recent findings highlight the potential of miRNAs as biomarkers to detect and predict progression of kidney diseases. Developing in parallel, novel methods for miRNA detection will facilitate the integration of these biomarkers into rapid routine clinical testing and existing care pathways. Validated kidney disease biomarkers also hold promise to identify novel therapeutic tools and targets. VIDEO ABSTRACT: http://links.lww.com/CONH/A43.

Proximity-induced superconductivity in epitaxial topological insulator/graphene/gallium heterostructures.

The introduction of superconductivity to the Dirac surface states of a topological insulator leads to a topological superconductor, which may support topological quantum computing through Majorana zero modes1,2. The development of a scalable material platform is key to the realization of topological quantum computing3,4. Here we report on the growth and properties of high-quality (Bi,Sb)2Te3/graphene/gallium heterostructures. Our synthetic approach enables atomically sharp layers at both hetero-interfaces, which in turn promotes proximity-induced superconductivity that originates in the gallium film. A lithography-free, van der Waals tunnel junction is developed to perform transport tunnelling spectroscopy. We find a robust, proximity-induced superconducting gap formed in the Dirac surface states in 5-10 quintuple-layer (Bi,Sb)2Te3/graphene/gallium heterostructures. The presence of a single Abrikosov vortex, where the Majorana zero modes are expected to reside, manifests in discrete conductance changes. The present material platform opens up opportunities for understanding and harnessing the application potential of topological superconductivity.

Analysis of Human Hyaluronan Synthase Gene Transcriptional Regulation and Downstream Hyaluronan Cell Surface Receptor Mobility in Myofibroblast Differentiation.

The ubiquitous extracellular glycosaminoglycan hyaluronan (HA) is a polymer 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. Emerging data continue to reveal functions attributable to HA in a variety of physiological and pathological contexts. Defining the mechanisms regulating expression of the human hyaluronan synthase (HAS) genes that encode the corresponding HA-synthesizing HAS enzymes is therefore important in the context of HA biology in health and disease. We describe here methods to analyze transcriptional regulation of the HAS and HAS2-antisense RNA 1 genes. Elucidation of mechanisms of HA interaction with receptors such as the cell surface molecule CD44 is also key to understanding HA function. To this end, we provide protocols for fluorescent recovery after photobleaching analysis of CD44 membrane dynamics in the process of fibroblast to myofibroblast differentiation, a phenotypic transition that is common to the pathology of fibrosis of large organs such as the liver and kidney.

MicroRNAs and their delivery in diabetic fibrosis.

The global prevalence of diabetes mellitus was estimated to be 463 million people in 2019 and is predicted to rise to 700 million by 2045. The associated financial and societal costs of this burgeoning epidemic demand an understanding of the pathology of this disease, and its complications, that will inform treatment to enable improved patient outcomes. Nearly two decades after the sequencing of the human genome, the significance of noncoding RNA expression is still being assessed. The family of functional noncoding RNAs known as microRNAs regulates the expression of most genes encoded by the human genome. Altered microRNA expression profiles have been observed both in diabetes and in diabetic complications. These transcripts therefore have significant potential and novelty as targets for therapy, therapeutic agents and biomarkers.

CD147 mediates the CD44s-dependent differentiation of myofibroblasts driven by transforming growth factor-ß1.

Progressive fibrosis leads to loss of organ function and affects many organs as a result of excessive extracellular matrix production. The ubiquitous matrix polysaccharide hyaluronan (HA) is central to this through association with its primary receptor, CD44, which exists as standard CD44 (CD44s) or multiple splice variants. Mediators such as profibrotic transforming growth factor (TGF)-ß1 and proinflammatory interleukin (IL)-1ß are widely associated with fibrotic progression. TGF-ß1 induces myofibroblast differentiation, while IL-1ß induces a proinflammatory fibroblast phenotype that promotes fibroblast binding to monocyte/macrophages. CD44 expression is essential for both responses. Potential CD44 splice variants involved, however, are unidentified. The TGF-ß1-activated CD44/epidermal growth factor receptor complex induces differentiation of metastatic cells through interactions with the matrix metalloproteinase inducer, CD147. This study aimed to determine the CD44 variants involved in TGF-ß1- and IL-1ß-mediated responses and to investigate the potential profibrotic role of CD147. Using immunocytochemistry and quantitative PCR, standard CD44s were shown to be essential for both TGF-ß1-induced fibroblast/myofibroblast differentiation and IL-1ß-induced monocyte binding. Co-immunoprecipitation identified that CD147 associated with CD44s. Using CD147-siRNA and confocal microscopy, we also determined that incorporation of the myofibroblast marker, αSMA, into F-actin stress fibers was prevented in the absence of CD147 and myofibroblast-dependent collagen gel contraction was inhibited. CD147 did not associate with HA, but removal of HA prevented the association of CD44s with CD147 at points of cell-cell contact. Taken together, our data suggest that CD44s/CD147 colocalization is essential in regulating the mechanical tension required for the αSMA incorporation into F-actin stress fibers that regulates myofibroblast phenotype.

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.

Single-Nucleus RNA Sequencing Identifies New Classes of Proximal Tubular Epithelial Cells in Kidney Fibrosis.

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. METHODS: 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. RESULTS: 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. CONCLUSIONS: These data identify unrecognized PTC phenotype heterogeneity and reveal novel PTCs associated with kidney fibrosis.

Determination of a microRNA signature of protective kidney ischemic preconditioning originating from proximal tubules.

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.

Unexpected Near-Infrared to Visible Nonlinear Optical Properties from 2-D Polar Metals.

Near-infrared-to-visible second harmonic generation from air-stable two-dimensional polar gallium and indium metals is described. The photonic properties of 2D metals, including the largest second-order susceptibilities reported for metals (approaching 10 nm/V), are determined by the atomic-level structure and bonding of two-to-three-atom-thick crystalline films. The bond character evolved from covalent to metallic over a few atomic layers, changing the out-of-plane metal-metal bond distances by approximately ten percent (0.2 Å), resulting in symmetry breaking and an axial electrostatic dipole that mediated the large nonlinear response. Two different orientations of the crystalline metal atoms, corresponding to lateral displacements <2 Å, persisted in separate micrometer-scale terraces to generate distinct harmonic polarizations. This strong atomic-level structure-property interplay suggests metal photonic properties can be controlled with atomic precision.

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.

Epitaxial graphene/silicon carbide intercalation: a minireview on graphene modulation and unique 2D materials.

Intercalation of atomic species through epitaxial graphene on silicon carbide began only a few years following its initial report in 2004. The impact of intercalation on the electronic properties of the graphene is well known; however, the intercalant itself can also exhibit intriguing properties not found in nature. This realization has inspired new interest in epitaxial graphene/silicon carbide (EG/SiC) intercalation, where the scope of the technique extends beyond modulation of graphene properties to the creation of new 2D forms of 3D materials. The mission of this minireview is to provide a concise introduction to EG/SiC intercalation and to demonstrate a simplified approach to EG/SiC intercalation. We summarize the primary techniques used to achieve and characterize EG/SiC intercalation, and show that thermal evaporation-based methods can effectively substitute for more complex synthesis techniques, enabling large-scale intercalation of non-refractory metals and compounds including two-dimensional silver (2D-Ag) and gallium nitride (2D-GaNx).

Neutrophil-derived miR-223 as local biomarker of bacterial peritonitis.

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.

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.

Biogenesis, Stabilization, and Transport of microRNAs in Kidney Health and Disease.

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.

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.

MicroRNAs as biomarkers in chronic kidney disease.

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. 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. 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.