Single-Cell RNA Sequencing Reveals Cardiac Fibroblast-Specific Transcriptomic Changes in Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is the most common cause of heart failure, with a complex aetiology involving multiple cell types. We aimed to detect cell-specific transcriptomic alterations in DCM through analysis that leveraged recent advancements in single-cell analytical tools. Single-cell RNA sequencing (scRNA-seq) data from human DCM cardiac tissue were subjected to an updated bioinformatic workflow in which unsupervised clustering was paired with reference label transfer to more comprehensively annotate the dataset. Differential gene expression was detected primarily in the cardiac fibroblast population. Bulk RNA sequencing was performed on an independent cohort of human cardiac tissue and compared with scRNA-seq gene alterations to generate a stratified list of higher-confidence, fibroblast-specific expression candidates for further validation. Concordant gene dysregulation was confirmed in TGFß-induced fibroblasts. Functional assessment of gene candidates showed that AEBP1 may play a significant role in fibroblast activation. This unbiased approach enabled improved resolution of cardiac cell-type-specific transcriptomic alterations in DCM.

Global analysis of Poales diversification - parallel evolution in space and time into open and closed habitats.

Poales are one of the most species-rich, ecologically and economically important orders of plants and often characterise open habitats, enabled by unique suites of traits. We test six hypotheses regarding the evolution and assembly of Poales in open and closed habitats throughout the world, and examine whether diversification patterns demonstrate parallel evolution. We sampled 42% of Poales species and obtained taxonomic and biogeographic data from the World Checklist of Vascular Plants database, which was combined with open/closed habitat data scored by taxonomic experts. A dated supertree of Poales was constructed. We integrated spatial phylogenetics with regionalisation analyses, historical biogeography and ancestral state estimations. Diversification in Poales and assembly of open and closed habitats result from dynamic evolutionary processes that vary across lineages, time and space, most prominently in tropical and southern latitudes. Our results reveal parallel and recurrent patterns of habitat and trait transitions in the species-rich families Poaceae and Cyperaceae. Smaller families display unique and often divergent evolutionary trajectories. The Poales have achieved global dominance via parallel evolution in open habitats, with notable, spatially and phylogenetically restricted divergences into strictly closed habitats.

Mapping the daily rhythmic transcriptome in the diabetic retina.

Retinal function changes dramatically from day to night, yet clinical diagnosis, treatments, and experimental sampling occur during the day. To begin to address this gap in our understanding of disease pathobiology, this study investigates whether diabetes affects the retina's daily rhythm of gene expression. Diabetic, Ins2Akita/J mice, and non-diabetic littermates were kept under a 12 h:12 h light/dark cycle until 4 months of age. mRNA sequencing was conducted in retinas collected every 4 h throughout the 24 hr light/dark cycle. Computational approaches were used to detect rhythmicity, predict acrophase, identify differential rhythmic patterns, analyze phase set enrichment, and predict upstream regulators. The retinal transcriptome exhibited a tightly regulated rhythmic expression with a clear 12-hr transcriptional axis. Day-peaking genes were enriched for DNA repair, RNA splicing, and ribosomal protein synthesis, night-peaking genes for metabolic processes and growth factor signaling. Although the 12-hr transcriptional axis is retained in the diabetic retina, it is phase advanced for some genes. Upstream regulator analysis for the phase-shifted genes identified oxygen-sensing mechanisms and HIF1alpha, but not the circadian clock, which remained in phase with the light/dark cycle. We propose a model in which, early in diabetes, the retina is subjected to an internal desynchrony with the circadian clock and its outputs are still light-entrained whereas metabolic pathways related to neuronal dysfunction and hypoxia are phase advanced. Further studies are now required to evaluate the chronic implications of such desynchronization on the development of diabetic retinopathy.

Zinc Supplementation Induced Transcriptional Changes in Primary Human Retinal Pigment Epithelium: A Single-Cell RNA Sequencing Study to Understand Age-Related Macular Degeneration.

Zinc supplementation has been shown to be beneficial to slow the progression of age-related macular degeneration (AMD). However, the molecular mechanism underpinning this benefit is not well understood. This study used single-cell RNA sequencing to identify transcriptomic changes induced by zinc supplementation. Human primary retinal pigment epithelial (RPE) cells could mature for up to 19 weeks. After 1 or 18 weeks in culture, we supplemented the culture medium with 125 µM added zinc for one week. RPE cells developed high transepithelial electrical resistance, extensive, but variable pigmentation, and deposited sub-RPE material similar to the hallmark lesions of AMD. Unsupervised cluster analysis of the combined transcriptome of the cells isolated after 2, 9, and 19 weeks in culture showed considerable heterogeneity. Clustering based on 234 pre-selected RPE-specific genes divided the cells into two distinct clusters, we defined as more and less differentiated cells. The proportion of more differentiated cells increased with time in culture, but appreciable numbers of cells remained less differentiated even at 19 weeks. Pseudotemporal ordering identified 537 genes that could be implicated in the dynamics of RPE cell differentiation (FDR < 0.05). Zinc treatment resulted in the differential expression of 281 of these genes (FDR < 0.05). These genes were associated with several biological pathways with modulation of ID1/ID3 transcriptional regulation. Overall, zinc had a multitude of effects on the RPE transcriptome, including several genes involved in pigmentation, complement regulation, mineralization, and cholesterol metabolism processes associated with AMD.

Nonmonotonic Superparamagnetic Behavior of the Ferritin Iron Core Revealed via Quantum Spin Relaxometry.

Ferritin is the primary storage protein in our body and is of significant interest in biochemistry, nanotechnology, and condensed matter physics. More specifically within this sphere of interest are the magnetic properties of the iron core of ferritin, which have been utilized as a contrast agent in applications such as magnetic resonance imaging. This magnetism depends on both the number of iron atoms present, L, and the nature of the magnetic ordering of their electron spins. In this work, we create a series of ferritin samples containing homogeneous iron loads and apply diamond-based quantum spin relaxometry to systematically study their room temperature magnetic properties. We observe anomalous magnetic behavior that can be explained using a theoretical model detailing a morphological change to the iron core occurring at relatively low iron loads. This model provides an L0.35±0.06 scaling of the uncompensated Fe spins, in agreement with previous theoretical predictions. The necessary inclusion of this morphological change within the model is also supported by electron microscopy studies of ferritin with low iron content. This provides evidence for a magnetic consequence of this morphological change and positions diamond-based quantum spin relaxometry as an effective, noninvasive tool for probing the magnetic properties of metalloproteins. The low detection limit (ferritin 2% loaded at a concentration of 7.5 ± 0.4 µg/mL) also makes this a promising method for precision applications where low analyte concentrations are unavoidable, such as in biological research or even clinical analysis.

Mesenchymal stromal cells-derived extracellular vesicles reprogramme macrophages in ARDS models through the miR-181a-5p-PTEN-pSTAT5-SOCS1 axis.

RATIONALE: A better understanding of the mechanism of action of mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) is needed to support their use as novel therapies for acute respiratory distress syndrome (ARDS). Macrophages are important mediators of ARDS inflammatory response. Suppressor of cytokine signalling (SOCS) proteins are key regulators of the macrophage phenotype switch. We therefore investigated whether SOCS proteins are involved in mediation of the MSC effect on human macrophage reprogramming. METHODS: Human monocyte-derived macrophages (MDMs) were stimulated with lipopolysaccharide (LPS) or plasma samples from patients with ARDS (these samples were previously classified into hypo-inflammatory and hyper-inflammatory phenotype) and treated with MSC conditioned medium (CM) or EVs. Protein expression was measured by Western blot. EV micro RNA (miRNA) content was determined by miRNA sequencing. In vivo: LPS-injured C57BL/6 mice were given EVs isolated from MSCs in which miR-181a had been silenced by miRNA inhibitor or overexpressed using miRNA mimic. RESULTS: EVs were the key component of MSC CM responsible for anti-inflammatory modulation of human macrophages. EVs significantly reduced secretion of tumour necrosis factor-α and interleukin-8 by LPS-stimulated or ARDS plasma-stimulated MDMs and this was dependent on SOCS1. Transfer of miR-181a in EVs downregulated phosphatase and tensin homolog (PTEN) and subsequently activated phosphorylated signal transducer and activator of transcription 5 (pSTAT5) leading to upregulation of SOCS1 in macrophages. In vivo, EVs alleviated lung injury and upregulated pSTAT5 and SOCS1 expression in alveolar macrophages in a miR181-dependent manner. Overexpression of miR-181a in MSCs significantly enhanced therapeutic efficacy of EVs in this model. CONCLUSION: miR-181a-PTEN-pSTAT5-SOCS1 axis is a novel pathway responsible for immunomodulatory effect of MSC EVs in ARDS.

Genome-Wide RNA Sequencing of Human Trabecular Meshwork Cells Treated with TGF-ß1: Relevance to Pseudoexfoliation Glaucoma.

Pseudoexfoliation glaucoma (XFG) is an aggressive form of secondary open angle glaucoma, characterised by the production of exfoliation material and is estimated to affect 30 million people worldwide. Activation of the TGF-ß pathway by TGF-ß1 has been implicated in the pathogenesis of pseudoexfoliation glaucoma. To further investigate the role of TGF-ß1 in glaucomatous changes in the trabecular meshwork (TM), we used RNA-Seq to determine TGF-ß1 induced changes in the transcriptome of normal human trabecular meshwork (HTM) cells. The main purpose of this study was to perform a hypothesis-independent RNA sequencing analysis to investigate genome-wide alterations in the transcriptome of normal HTMs stimulated with TGF-ß1 and investigate possible pathophysiological mechanisms driving XFG. Our results identified multiple differentially expressed genes including several genes known to be present in exfoliation material. Significantly altered pathways, biological processes and molecular functions included extracellular matrix remodelling, Hippo and Wnt pathways, the unfolded protein response, oxidative stress, and the antioxidant system. This cellular model of pseudoexfoliation glaucoma can provide insight into disease pathogenesis and support the development of novel therapeutic interventions.

Genome-wide transcriptome profiling of human trabecular meshwork cells treated with TGF-ß2.

Glaucoma is a complex neurodegenerative disease resulting in progressive optic neuropathy and is a leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG) is the predominant form affecting 65.5 million people globally. Despite the prevalence of POAG and the identification of over 120 glaucoma related genetic loci, the underlaying molecular mechanisms are still poorly understood. The transforming growth factor beta (TGF-ß) signalling pathway is implicated in the molecular pathology of POAG. To gain a better understanding of the role TGF-ß2 plays in the glaucomatous changes to the molecular pathology in the trabecular meshwork, we employed RNA-Seq to delineate the TGF-ß2 induced changes in the transcriptome of normal primary human trabecular meshwork cells (HTM). We identified a significant number of differentially expressed genes and associated pathways that contribute to the pathogenesis of POAG. The differentially expressed genes were predominantly enriched in ECM regulation, TGF-ß signalling, proliferation/apoptosis, inflammation/wound healing, MAPK signalling, oxidative stress and RHO signalling. Canonical pathway analysis confirmed the enrichment of RhoA signalling, inflammatory-related processes, ECM and cytoskeletal organisation in HTM cells in response to TGF-ß2. We also identified novel genes and pathways that were affected after TGF-ß2 treatment in the HTM, suggesting additional pathways are activated, including Nrf2, PI3K-Akt, MAPK and HIPPO signalling pathways. The identification and characterisation of TGF-ß2 dependent differentially expressed genes and pathways in HTM cells is essential to understand the patho-physiology of glaucoma and to develop new therapeutic agents.

Short and long-term effect of dexamethasone on the transcriptome profile of primary human trabecular meshwork cells in vitro.

In the quest of identifying newer molecular targets for the management of glucocorticoid-induced ocular hypertension (GC-OHT) and glaucoma (GCG), several microarray studies have attempted to investigate the genome-wide transcriptome profiling of primary human trabecular meshwork (TM) cells in response to dexamethasone (DEX). However, no studies are reported so far to demonstrate the temporal changes in the expression of genes in the cultured human TM cells in response to DEX treatment. Therefore, in the present study, the time-dependent changes in the genome-wide expression of genes in primary human TM cells after short (16 hours: 16 h) and long exposure (7 days: 7 d) of DEX was investigated using RNA sequencing. There were 199 (118 up-regulated; 81 down-regulated) and 525 (119 up-regulated; 406 down-regulated) DEGs in 16 h and 7 d treatment groups respectively. The unique genes identified in 16 h and 7 d treatment groups were 152 and 478 respectively. This study found a distinct gene signature and pathways between two treatment regimes. Longer exposure of DEX treatment showed a dys-regulation of Wnt and Rap1 signaling and so highlighted potential therapeutic targets for pharmacological management of GC-OHT/glaucoma.

Mini-XT, a miniaturized tagmentation-based protocol for efficient sequencing of SARS-CoV-2.

BACKGROUND: The COVID-19 pandemic has highlighted the importance of whole genome sequencing (WGS) of SARS-CoV-2 to inform public health policy. By enabling definition of lineages it facilitates tracking of the global spread of the virus. The evolution of new variants can be monitored and knowledge of specific mutations provides insights into the mechanisms through which the virus increases transmissibility or evades immunity. To date almost 1 million SARS-CoV-2 genomes have been sequenced by members of the COVID-19 Genomics UK (COG-UK) Consortium. To achieve similar feats in a more cost-effective and sustainable manner in future, improved high throughput virus sequencing protocols are required. We have therefore developed a miniaturized library preparation protocol with drastically reduced consumable use and costs. RESULTS: We present the 'Mini-XT' miniaturized tagmentation-based library preparation protocol available on protocols.io ( https://doi.org/10.17504/protocols.io.bvntn5en ). SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 multiplex RT-PCR protocol and purified using a conventional liquid handling system. Acoustic liquid transfer (Echo 525) was employed to reduce reaction volumes and the number of tips required for a Nextera XT library preparation. Sequencing was performed on an Illumina MiSeq. The final version of Mini-XT has been used to sequence 4384 SARS-CoV-2 samples from N. Ireland with a COG-UK QC pass rate of 97.4%. Sequencing quality was comparable and lineage calling consistent for replicate samples processed with full volume Nextera DNA Flex (333 samples) or using nanopore technology (20 samples). SNP calling between Mini-XT and these technologies was consistent and sequences from replicate samples paired together in maximum likelihood phylogenetic trees. CONCLUSIONS: The Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes eightfold and halving overall tip usage from sample to sequence to provide concomitant cost savings relative to standard protocols. This will enable more efficient high-throughput sequencing of SARS-CoV-2 isolates and future pathogen WGS.

Acoustomicrofluidic Concentration and Signal Enhancement of Fluorescent Nanodiamond Sensors.

Diamond nitrogen-vacancy (NV) centers constitute a promising class of quantum nanosensors owing to the unique magneto-optic properties associated with their spin states. The large surface area and photostability of diamond nanoparticles, together with their relatively low synthesis costs, make them a suitable platform for the detection of biologically relevant quantities such as paramagnetic ions and molecules in solution. Nevertheless, their sensing performance in solution is often hampered by poor signal-to-noise ratios and long acquisition times due to distribution inhomogeneities throughout the analyte sample. By concentrating the diamond nanoparticles through an intense microcentrifugation effect in an acoustomicrofluidic device, we show that the resultant dense NV ensembles within the diamond nanoparticles give rise to an order-of-magnitude improvement in the measured acquisition time. The ability to concentrate nanoparticles under surface acoustic wave (SAW) microcentrifugation in a sessile droplet is, in itself, surprising given the well-documented challenge of achieving such an effect for particles below 1 µm in dimension. In addition to a demonstration of their sensing performance, we thus reveal in this work that the reason why the diamond nanoparticles readily concentrate under the SAW-driven recirculatory flow can be attributed to their considerably higher density and hence larger acoustic contrast compared to those for typical particles and cells for which the SAW microcentrifugation flow has been shown to date.

Quantum magnetic imaging of iron organelles within the pigeon cochlea.

The ability of pigeons to sense geomagnetic fields has been conclusively established despite a notable lack of determination of the underlying biophysical mechanisms. Quasi-spherical iron organelles previously termed "cuticulosomes" in the cochlea of pigeons have potential relevance to magnetoreception due to their location and iron composition; however, data regarding the magnetic susceptibility of these structures are currently limited. Here quantum magnetic imaging techniques are applied to characterize the magnetic properties of individual iron cuticulosomes in situ. The stray magnetic fields emanating from cuticulosomes are mapped and compared to a detailed analytical model to provide an estimate of the magnetic susceptibility of the individual particles. The images reveal the presence of superparamagnetic and ferrimagnetic domains within individual cuticulosomes and magnetic susceptibilities within the range 0.029 to 0.22. These results provide insights into the elusive physiological roles of cuticulosomes. The susceptibilities measured are not consistent with a torque-based model of magnetoreception, placing iron storage and stereocilia stabilization as the two leading putative cuticulosome functions. This work establishes quantum magnetic imaging as an important tool to complement the existing array of techniques used to screen for potential magnetic particle-based magnetoreceptor candidates.

Preferential coupling of diamond NV centres in step-index fibres.

Diamonds containing the negatively charged nitrogen-vacancy centre are a promising system for room-temperature magnetometry. The combination of nano- and micro-diamond particles with optical fibres provides an option for deploying nitrogen-vacancy magnetometers in harsh and challenging environments. Here we numerically explore the coupling efficiency from nitrogen-vacancy centres within a diamond doped at the core/clad interface across a range of commercially available fibre types so as to inform the design process for a diamond in fibre magnetometers. We determine coupling efficiencies from nitrogen-vacancy centres to the guided modes of a step-index fibre and predict the optically detected magnetic resonance (ODMR) generated by a ensemble of four nitrogen-vacancy centres in this hybrid fibre system. Our results show that the coupling efficiency is enhanced with a high refractive index difference between the fibre core and cladding and depends on the radial position of the nitrogen-vacancy centres in the fibre core. Our ODMR simulations show that due to the preferential coupling of the nitrogen-vacancy emission to the fibre guided modes, certain magnetometry features such as ODMR contrast can be enhanced and lead to improved sensitivity in such diamond-fibre systems, relative to conventional diamond only ensemble geometries.

Re-examining ferritin-bound iron: current and developing clinical tools.

Iron is a highly important metal ion cofactor within the human body, necessary for haemoglobin synthesis, and required by a wide range of enzymes for essential metabolic processes. Iron deficiency and overload both pose significant health concerns and are relatively common world-wide health hazards. Effective measurement of total iron stores is a primary tool for both identifying abnormal iron levels and tracking changes in clinical settings. Population based data is also essential for tracking nutritional trends. This review article provides an overview of the strengths and limitations associated with current techniques for diagnosing iron status, which sets a basis to discuss the potential of a new serum marker - ferritin-bound iron - and the improvement it could offer to iron assessment.

Single-cell transcriptomic profiling provides insights into retinal endothelial barrier properties.

Purpose: To better characterize retinal endothelial barrier properties through analysis of individual transcriptomes of primary bovine retinal microvascular endothelial cells (RMECs). Methods: Individual RMECs were captured on the Fluidigm C1 system, cDNA libraries were prepared using a Nextera XT kit, and sequencing was performed on a NextSeq system (Illumina). Data analysis was performed using R packages Scater, SC3, and Seurat, and the browser application Automated Single-cell Analysis Pipeline (ASAP). Alternative splicing events in single cells were quantified with Outrigger. Cytoscape was used for network analyses. Results: Application of a single-cell RNA sequencing (scRNA-seq) analysis workflow showed that RMECs form a relatively homogeneous population in culture, with the main differences related to proliferation status. Expression of markers from along the arteriovenous tree suggested that most cells originated from capillaries. Average gene expression levels across all cells were used to develop an in silico model of the inner blood-retina barrier incorporating junctional proteins not previously reported within the retinal vasculature. Correlation of barrier gene expression among individual cells revealed a subgroup of genes highly correlated with PECAM-1 at the center of the correlation network. Numerous alternative splicing events involving exons within microvascular barrier genes were observed, and in many cases, individual cells expressed one isoform exclusively. Conclusions: We optimized a workflow for single-cell transcriptomics in primary RMECs. The results provide fundamental insights into the genes involved in formation of the retinal-microvascular barrier.

Phexpo: a package for bidirectional enrichment analysis of phenotypes and chemicals.

Phenotypes are the result of the complex interplay between environmental and genetic factors. To better understand the interactions between chemical compounds and human phenotypes, and further exposome research we have developed "phexpo," a tool to perform and explore bidirectional chemical and phenotype interactions using enrichment analyses. Phexpo utilizes gene annotations from 2 curated public repositories, the Comparative Toxicogenomics Database and the Human Phenotype Ontology. We have applied phexpo in 3 case studies linking: (1) individual chemicals (a drug, warfarin, and an industrial chemical, chloroform) with phenotypes, (2) individual phenotypes (left ventricular dysfunction) with chemicals, and (3) multiple phenotypes (covering polycystic ovary syndrome) with chemicals. The results of these analyses demonstrated successful identification of relevant chemicals or phenotypes supported by bibliographic references. The phexpo R package (https://github.com/GHLCLab/phexpo) provides a new bidirectional analyses approach covering relationships from chemicals to phenotypes and from phenotypes to chemicals.

Enhanced Widefield Quantum Sensing with Nitrogen-Vacancy Ensembles Using Diamond Nanopillar Arrays.

Surface micro- and nano-patterning techniques are often employed to enhance the optical interface to single photoluminescent emitters in diamond, but the utility of such surface structuring in applications requiring ensembles of emitters is still open to investigation. Here, we demonstrate scalable and fault-tolerant fabrication of closely packed arrays of fluorescent diamond nanopillars, each hosting its own dense, uniformly bright ensemble of near-surface nitrogen-vacancy centers. We explore the optimal sizes for these structures and realize enhanced spin and photoluminescence properties resulting in a 4.5 times increase in optically detected magnetic resonance sensitivity when compared to unpatterned surfaces. Utilizing the increased measurement sensitivity, we image the mechanical stress tensor in each diamond pillar across the arrays and show that the fabrication process has a negligible impact on in-built stress compared to the unpatterned surface. Our results represent a valuable pathway toward future multimodal and vector-resolved imaging studies, for instance in biological contexts.

Pathways, Processes, and Candidate Drugs Associated with a Hoxa Cluster-Dependency Model of Leukemia.

High expression of the HOXA cluster correlates with poor clinical outcome in acute myeloid leukemias, particularly those harboring rearrangements of the mixed-lineage-leukemia gene (MLLr). Whilst decreased HOXA expression acts as a readout for candidate experimental therapies, the necessity of the HOXA cluster for leukemia maintenance has not been fully explored. Primary leukemias were generated in hematopoietic stem/progenitor cells from Cre responsive transgenic mice for conditional deletion of the Hoxa locus. Hoxa deletion resulted in reduced proliferation and colony formation in which surviving leukemic cells retained at least one copy of the Hoxa cluster, indicating dependency. Comparative transcriptome analysis of Hoxa wild type and deleted leukemic cells identified a unique gene signature associated with key pathways including transcriptional mis-regulation in cancer, the Fanconi anemia pathway and cell cycle progression. Further bioinformatics analysis of the gene signature identified a number of candidate FDA-approved drugs for potential repurposing in high HOXA expressing cancers including MLLr leukemias. Together these findings support dependency for an MLLr leukemia on Hoxa expression and identified candidate drugs for further therapeutic evaluation.

Differential Expression of Urinary Exosomal MicroRNAs miR-21-5p and miR-30b-5p in Individuals with Diabetic Kidney Disease.

Biomarkers for the identification of diabetic kidney disease (DKD) are needed as current tests lack sensitivity for detecting early kidney damage. MicroRNAs (miRNAs) are short, non-coding regulatory ribonucleic acid (RNA) molecules commonly found in urinary exosomes differentially expressed as renal function declines. We evaluated urinary exosomal miRNA expression in persons with type 2 diabetes mellitus and DKD (T2DKD). 87 human urinary exosomal miRNAs were profiled in a discovery cohort of patients with T2DKD (n = 14) and age and gender matched controls with type 2 diabetes mellitus and normal renal function (T2DNRF; n = 15). Independent validation of differentially expressed target miRNAs was performed in a second cohort with T2DKD (n = 22) and two control groups: T2DNRF (n = 15) and controls with chronic kidney disease (CCKD) and poor renal function without diabetes (n = 18). In the discovery cohort, urinary miR-21-5p, let-7e-5p and miR-23b-3p were significantly upregulated in T2DKD compared to T2DNRF (p < 0.05). Conversely, miR-30b-5p and miR-125b-5p expression was significantly lower in T2DKD (p < 0.05). Independent validation confirmed up-regulation of miR-21-5p in the replication cohort in T2DKD (2.13-fold, p = 0.006) and in CCKD (1.73-fold, p = 0.024). In contrast, miR-30b-5p was downregulated in T2DKD (0.82-fold, p = 0.006) and in CCKD (0.66-fold, p < 0.002). This study identified differential expression of miR-21-5p and miR-30b-5p in individuals with diabetic kidney disease and poor renal function. These miRNAs represent potential biomarkers associated with the pathogenesis of renal dysfunction.