The single-cell transcriptomic landscape of early human diabetic nephropathy

Diabetic nephropathy is characterized by damage to both the glomerulus and tubulointerstitium, but relatively little is known about accompanying cell-specific changes in gene expression. We performed unbiased single-nucleus RNA sequencing (snRNA-seq) on cryopreserved human diabetic kidney samples to generate 23,980 single-nucleus transcriptomes from 3 control and 3 early diabetic nephropathy samples. All major cell types of the kidney were represented in the final dataset. Side-by-side comparison demonstrated cell-type–specific changes in gene expression that are important for ion transport, angiogenesis, and immune cell activation. In particular, we show that the diabetic thick ascending limb, late distal convoluted tubule, and principal cells all adopt a gene expression signature consistent with increased potassium secretion, including alterations in Na+/K+-ATPase, WNK1, mineralocorticoid receptor, and NEDD4L expression, as well as decreased paracellular calcium and magnesium reabsorption. We also identify strong angiogenic signatures in glomerular cell types, proximal convoluted tubule, distal convoluted tubule, and principal cells. Taken together, these results suggest that increased potassium secretion and angiogenic signaling represent early kidney responses in human diabetic nephropathy.

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The Single Cell Transcriptomic Landscape of Early Human Diabetic Nephropathy

10.1101/645424 ◽

2019 ◽

Cited By ~ 1

Author(s):

Parker C. Wilson ◽

Haojia Wu ◽

Yuhei Kirita ◽

Kohei Uchimura ◽

Helmut G. Rennke ◽

...

Keyword(s):

Gene Expression ◽

Diabetic Nephropathy ◽

Rna Sequencing ◽

Cell Types ◽

Distal Convoluted Tubule ◽

Principal Cells ◽

Single Nucleus ◽

Potassium Secretion ◽

Calcium And Magnesium ◽

Early Diabetic Nephropathy

AbstractDiabetic nephropathy is characterized by damage to both the glomerulus and tubulointerstitium, but relatively little is known about accompanying cell-specific changes in gene expression. We performed unbiased single nucleus RNA sequencing (snRNAseq) on cryopreserved human diabetic kidney samples to generate 23,980 single nucleus transcriptomes from three control and three early diabetic nephropathy samples. All major cell types of the kidney were represented in the final dataset. Side by side comparison demonstrated cell-type-specific changes in gene expression that are important for ion transport, angiogenesis, and immune cell activation. In particular, we show that the diabetic loop of Henle, late distal convoluted tubule, and principal cells all adopt a gene expression signature consistent with increased potassium secretion, including alterations in Na-K+-ATPase, WNK1, mineralocorticoid receptor and NEDD4L expression, as well as decreased paracellular calcium and magnesium reabsorption. We also identify strong angiogenic signatures in glomerular cell types, proximal convoluted tubule, distal convoluted tubule and principal cells. Taken together, these results suggest that increased potassium secretion and angiogenic signaling represent early kidney responses in human diabetic nephropathy.Significance StatementSingle nucleus RNA sequencing revealed gene expression changes in early diabetic nephropathy that promote urinary potassium secretion and decreased calcium and magnesium reabsorption. Multiple cell types exhibited angiogenic signatures, which may represent early signs of aberrant angiogenesis. These alterations may help to identify biomarkers for disease progression or signaling pathways amenable to early intervention.

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Single-cell RNA sequencing reveals the mesangial identity and species diversity of glomerular cell transcriptomes

Nature Communications ◽

10.1038/s41467-021-22331-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Bing He ◽

Ping Chen ◽

Sonia Zambrano ◽

Dina Dabaghie ◽

Yizhou Hu ◽

...

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Human Kidney ◽

Cell Types ◽

Model Organisms ◽

Mural Cell ◽

Glomerular Cell ◽

Individual Gene ◽

The Individual

AbstractMolecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), are missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we use Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis reveals genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells. Importantly, we detect heterogeneity in glomerulus-associated Pdgfrb-expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observe remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies.

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Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM

10.1101/786285 ◽

2019 ◽

Cited By ~ 4

Author(s):

Marcus Alvarez ◽

Elior Rahmani ◽

Brandon Jew ◽

Kristina M. Garske ◽

Zong Miao ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cell Types ◽

Supervised Machine Learning ◽

Data Sets ◽

Rna Seq ◽

Novel Approach ◽

Single Nucleus ◽

Downstream Analysis

AbstractSingle-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. Contrary to single-cell RNA seq (scRNA-seq), we observe that snRNA-seq is commonly subject to contamination by high amounts of extranuclear background RNA, which can lead to identification of spurious cell types in downstream clustering analyses if overlooked. We present a novel approach to remove debris-contaminated droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: 1) human differentiating preadipocytes in vitro, 2) fresh mouse brain tissue, and 3) human frozen adipose tissue (AT) from six individuals. All three data sets showed various degrees of extranuclear RNA contamination. We observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq data, we also successfully applied DIEM to single-cell data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

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A mathematical model of rat distal convoluted tubule. II. Potassium secretion along the connecting segment

AJP Renal Physiology ◽

10.1152/ajprenal.00044.2005 ◽

2005 ◽

Vol 289 (4) ◽

pp. F721-F741 ◽

Cited By ~ 37

Author(s):

Alan M. Weinstein

Keyword(s):

Water Permeability ◽

Collecting Duct ◽

Cell Types ◽

Distal Convoluted Tubule ◽

Cortical Collecting Duct ◽

Transport Activity ◽

Luminal Membrane ◽

In Series ◽

Potassium Secretion ◽

Osmotic Equilibration

A simulation of the rat distal convoluted tubule (DCT) is completed with a model of the late portion, or connecting tubule (CNT). This CNT model is developed by relying on a prior cortical collecting duct (CCD) model (Weinstein AM. Am J Physiol Renal Physiol 280: F1072–F1092, 2001), and scaling up transport activity of the three cell types to a level appropriate for DCT. The major difference between the two tubule segments is the lower CNT water permeability. In early CNT the luminal solution is hypotonic, with a K+ concentration less than that of plasma, and it is predicted that osmotic equilibration requires the whole length of CNT, to end with a nearly isotonic fluid, whose K+ concentration is severalfold greater than plasma. With respect to potassium secretion, early CNT conditions are conducive to maximal fluxes, whereas late conditions require the capacity to transport against a steep electrochemical gradient. The parameter dependence for K+ secretion under each condition is different: maximal secretion depends on luminal membrane K+ permeability, but the limiting luminal K+ concentration does not. However, maximal secretion and the limiting gradient are both enhanced by greater Na+ reabsorption. While higher CNT water permeability depresses K+ secretion, it favors Na+ reabsorption. Thus in antidiuresis there is a trade-off between enhanced Na+-dependent K+ secretion and the attenuation of K+ secretion by slow flow. When the CNT model is configured in series with the early DCT, thiazide diuretics promote renal K+ wasting by shifting Na+ reabsorption from early DCT to CNT; they promote alkalosis by shifting the remaining early DCT Na+ reabsorption to Na+/H+ exchange. This full DCT is suitable for simulating the defects of hyperkalemic hypertension, but the model offers no suggestion of a tight junction abnormality that might contribute to the phenotype.

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A Single-Cell Transcriptome Atlas of the Mouse Glomerulus

Journal of the American Society of Nephrology ◽

10.1681/asn.2018030238 ◽

2018 ◽

Vol 29 (8) ◽

pp. 2060-2068 ◽

Cited By ~ 53

Author(s):

Nikos Karaiskos ◽

Mahdieh Rahmatollahi ◽

Anastasiya Boltengagen ◽

Haiyue Liu ◽

Martin Hoehne ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Single Cell ◽

Mesangial Cells ◽

Transcriptional Profiling ◽

Wild Type Mouse ◽

Cell Types ◽

Cellular Heterogeneity ◽

Marker Genes ◽

Glomerular Cell

Background Three different cell types constitute the glomerular filter: mesangial cells, endothelial cells, and podocytes. However, to what extent cellular heterogeneity exists within healthy glomerular cell populations remains unknown.Methods We used nanodroplet-based highly parallel transcriptional profiling to characterize the cellular content of purified wild-type mouse glomeruli.Results Unsupervised clustering of nearly 13,000 single-cell transcriptomes identified the three known glomerular cell types. We provide a comprehensive online atlas of gene expression in glomerular cells that can be queried and visualized using an interactive and freely available database. Novel marker genes for all glomerular cell types were identified and supported by immunohistochemistry images obtained from the Human Protein Atlas. Subclustering of endothelial cells revealed a subset of endothelium that expressed marker genes related to endothelial proliferation. By comparison, the podocyte population appeared more homogeneous but contained three smaller, previously unknown subpopulations.Conclusions Our study comprehensively characterized gene expression in individual glomerular cells and sets the stage for the dissection of glomerular function at the single-cell level in health and disease.

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Targeting MyD88 Downregulates Inflammatory Mediators and Pathogenic Processes in PBMC From DMARDs-Naïve Rheumatoid Arthritis Patients

Frontiers in Pharmacology ◽

10.3389/fphar.2021.800220 ◽

2021 ◽

Vol 12 ◽

Author(s):

Sergio Ramirez-Perez ◽

Edith Oregon-Romero ◽

Itzel Viridiana Reyes-Perez ◽

Pallavi Bhattaram

Keyword(s):

Rheumatoid Arthritis ◽

Gene Expression ◽

Inflammatory Mediators ◽

Mononuclear Cells ◽

Therapeutic Potential ◽

Gene Expression Signature ◽

Cell Types ◽

Cell Receptor ◽

Matrix Metalloproteases ◽

Peripheral Blood Mononuclear

MyD88-dependent intracellular signalling cascades and subsequently NF-kappaB-mediated transcription lead to the dynamic inflammatory processes underlying the pathogenesis of rheumatoid arthritis (RA) and related autoimmune diseases. This study aimed to identify the effect of the MyD88 dimerization inhibitor, ST2825, as a modulator of pathogenic gene expression signatures and systemic inflammation in disease-modifying antirheumatic drugs (DMARDs)-naïve RA patients. We analyzed bulk RNA-seq from peripheral blood mononuclear cells (PBMC) in DMARDs-naïve RA patients after stimulation with LPS and IL-1β. The transcriptional profiles of ST2825-treated PBMC were analyzed to identify its therapeutic potential. Ingenuity Pathway Analysis was implemented to identify downregulated pathogenic processes. Our analysis revealed 631 differentially expressed genes between DMARDs-naïve RA patients before and after ST2825 treatment. ST2825-treated RA PBMC exhibited a gene expression signature similar to that of healthy controls PBMC by downregulating the expression of proinflammatory cytokines, chemokines and matrix metalloproteases. In addition, B cell receptor, IL-17 and IL-15 signalling were critically downregulated pathways by ST2825. Furthermore, we identified eight genes (MMP9, CXCL9, MZB1, FUT7, TGM2, IGLV1-51, LINC01010, and CDK1) involved in pathogenic processes that ST2825 can potentially inhibit in distinct cell types within the RA synovium. Overall, our findings indicate that targeting MyD88 effectively downregulates systemic inflammatory mediators and modulates the pathogenic processes in PBMC from DMARDs-naïve RA patients. ST2825 could also potentially inhibit upregulated genes in the RA synovium, preventing synovitis and joint degeneration.

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Single-nucleus RNA sequencing shows convergent evidence from different cell types for altered synaptic plasticity in major depressive disorder

10.1101/384479 ◽

2018 ◽

Cited By ~ 7

Author(s):

Corina Nagy ◽

Malosree Maitra ◽

Arnaud Tanti ◽

Matthew Suderman ◽

Jean-Francois Théroux ◽

...

Keyword(s):

Gene Expression ◽

Major Depressive Disorder ◽

Synaptic Plasticity ◽

Depressive Disorder ◽

Cell Types ◽

Specific Cell ◽

Major Depressive ◽

Differential Correlation ◽

Dorsolateral Prefrontal ◽

Single Nucleus

AbstractMajor depressive disorder (MDD) is a complex illness that involves the interaction of different brain systems, pathways, and cell types. Past molecular studies of MDD relied on cellular homogenates of post-mortem brain tissue, making it impossible to determine gene expression changes within individual cells. Using single-cell transcriptomics, we examined almost 80,000 nuclei from the dorsolateral prefrontal cortex of individuals with MDD and healthy controls. Our analyses identified 26 distinct cellular clusters, and over 60% of these showed transcriptional differences between groups. Specifically, 96 genes were differentially expressed, the majority of which were downregulated. Convergent evidence from our analyses, including gene expression, differential correlation, and gene ontology implicated dysregulation of synaptic plasticity in the etiopathogenesis of MDD. Our results show that this high-resolution approach can reveal previously undetectable changes in specific cell types in the context of complex phenotypes and heterogeneous tissues.

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Molecular Assessment of Epiretinal Membrane: Activated Microglia, Oxidative Stress and Inflammation

Antioxidants ◽

10.3390/antiox9080654 ◽

2020 ◽

Vol 9 (8) ◽

pp. 654 ◽

Cited By ~ 1

Author(s):

Sushma Vishwakarma ◽

Rishikesh Kumar Gupta ◽

Saumya Jakati ◽

Mudit Tyagi ◽

Rajeev Reddy Pappuru ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Epiretinal Membrane ◽

Hypoxia Inducible Factor ◽

Gene Expression Signature ◽

Cell Types ◽

Activated Microglia ◽

Inflammatory Conditions ◽

New Information ◽

And Function

Fibrocellular membrane or epiretinal membrane (ERM) forms on the surface of the inner limiting membrane (ILM) in the inner retina and alters the structure and function of the retina. ERM formation is frequently observed in ocular inflammatory conditions, such as proliferative diabetic retinopathy (PDR) and retinal detachment (RD). Although peeling of the ERM is used as a surgical intervention, it can inadvertently distort the retina. Our goal is to design alternative strategies to tackle ERMs. As a first step, we sought to determine the composition of the ERMs by identifying the constituent cell-types and gene expression signature in patient samples. Using ultrastructural microscopy and immunofluorescence analyses, we found activated microglia, astrocytes, and Müller glia in the ERMs from PDR and RD patients. Moreover, oxidative stress and inflammation associated gene expression was significantly higher in the RD and PDR membranes as compared to the macular hole samples, which are not associated with inflammation. We specifically detected differential expression of hypoxia inducible factor 1-α (HIF1-α), proinflammatory cytokines, and Notch, Wnt, and ERK signaling pathway-associated genes in the RD and PDR samples. Taken together, our results provide new information to potentially develop methods to tackle ERM formation.

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Graded regulation of cellular quiescence depth between proliferation and senescence by a lysosomal dimmer switch

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915905116 ◽

2019 ◽

Vol 116 (45) ◽

pp. 22624-22634 ◽

Cited By ~ 9

Author(s):

Kotaro Fujimaki ◽

Ruoyan Li ◽

Hengyu Chen ◽

Kimiko Della Croce ◽

Hao Helen Zhang ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Signature ◽

Cell Types ◽

The Body ◽

Cellular Mechanisms ◽

Lysosomal Function ◽

Quiescent Cells ◽

Common Transition

The reactivation of quiescent cells to proliferate is fundamental to tissue repair and homeostasis in the body. Often referred to as the G0 state, quiescence is, however, not a uniform state but with graded depth. Shallow quiescent cells exhibit a higher tendency to revert to proliferation than deep quiescent cells, while deep quiescent cells are still fully reversible under physiological conditions, distinct from senescent cells. Cellular mechanisms underlying the control of quiescence depth and the connection between quiescence and senescence are poorly characterized, representing a missing link in our understanding of tissue homeostasis and regeneration. Here we measured transcriptome changes as rat embryonic fibroblasts moved from shallow to deep quiescence over time in the absence of growth signals. We found that lysosomal gene expression was significantly up-regulated in deep quiescence, and partially compensated for gradually reduced autophagy flux. Reducing lysosomal function drove cells progressively deeper into quiescence and eventually into a senescence-like irreversibly arrested state; increasing lysosomal function, by lowering oxidative stress, progressively pushed cells into shallower quiescence. That is, lysosomal function modulates graded quiescence depth between proliferation and senescence as a dimmer switch. Finally, we found that a gene-expression signature developed by comparing deep and shallow quiescence in fibroblasts can correctly classify a wide array of senescent and aging cell types in vitro and in vivo, suggesting that while quiescence is generally considered to protect cells from irreversible arrest of senescence, quiescence deepening likely represents a common transition path from cell proliferation to senescence, related to aging.

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Single-nucleus RNA sequencing of mouse auditory cortex reveals critical period triggers and brakes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1920433117 ◽

2020 ◽

Vol 117 (21) ◽

pp. 11744-11752 ◽

Cited By ~ 6

Author(s):

Brian T. Kalish ◽

Tania R. Barkat ◽

Erin E. Diel ◽

Elizabeth J. Zhang ◽

Michael E. Greenberg ◽

...

Keyword(s):

Gene Expression ◽

Auditory Cortex ◽

Rna Sequencing ◽

Critical Period ◽

Neural Circuit ◽

Developmental Process ◽

Brain Plasticity ◽

Cell Types ◽

Altered Expression ◽

Single Nucleus

Auditory experience drives neural circuit refinement during windows of heightened brain plasticity, but little is known about the genetic regulation of this developmental process. The primary auditory cortex (A1) of mice exhibits a critical period for thalamocortical connectivity between postnatal days P12 and P15, during which tone exposure alters the tonotopic topography of A1. We hypothesized that a coordinated, multicellular transcriptional program governs this window for patterning of the auditory cortex. To generate a robust multicellular map of gene expression, we performed droplet-based, single-nucleus RNA sequencing (snRNA-seq) of A1 across three developmental time points (P10, P15, and P20) spanning the tonotopic critical period. We also tone-reared mice (7 kHz pips) during the 3-d critical period and collected A1 at P15 and P20. We identified and profiled both neuronal (glutamatergic and GABAergic) and nonneuronal (oligodendrocytes, microglia, astrocytes, and endothelial) cell types. By comparing normal- and tone-reared mice, we found hundreds of genes across cell types showing altered expression as a result of sensory manipulation during the critical period. Functional voltage-sensitive dye imaging confirmed GABA circuit function determines critical period onset, while Nogo receptor signaling is required for its closure. We further uncovered previously unknown effects of developmental tone exposure on trajectories of gene expression in interneurons, as well as candidate genes that might execute tonotopic plasticity. Our single-nucleus transcriptomic resource of developing auditory cortex is thus a powerful discovery platform with which to identify mediators of tonotopic plasticity.

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