Biology subjects

Akilesh, S.

Publications and source records attributed to Akilesh, S..

10 recordsLinked to original sources

bioRxiv2026

A Human Precision Cut Kidney Slice Platform for Studying Glomerular Biology and Therapeutic Responses

Introduction: Short term human precision cut kidney slice cultures (PCKS) represent a tractable experimental that contains mature kidney cell types within their native anatomic context and that can be manipulated in vitro. In this study, we employ genomic tools to analyze the PCKS response to culture conditions and their suitability for study of glomerular disease biology. Methods: The impact of culture medium and oxygenation on PCKS viability and gene expression was explored using spatial transcriptomics and traditional histologic approaches. The biological response of PCKS to IFN{gamma} stimulation and signaling suppression by the JAK1/2 inhibitor baricitinib were also explored. Results: A genome wide atlas of glomerular and tubulointerstitial gene expression patterns was generated from PCKS cultured in different media and oxygenation conditions and at various timepoints in culture. IFN{gamma} induced APOL1 gene and protein expression in glomeruli, which could be suppressed by baricitinib. Expression changes were reproducible in PCKS derived from multiple donors. Conclusions: PCKS are reproducibly responsive to IFN{gamma} stimulation, which will allow dissection of APOL1 mediated glomerular disease mechanisms in a human tissue model system. Spatial transcriptomic profiling of tissue responses helps to tailor culture medium/oxygenation to optimize the PCKS system to address specific biological questions.

cell biology↗

bioRxiv2026

CD19 CAR Engineered T Regulatory Cells Promote Durable Immune Tolerance in Experimental Systemic Lupus Erythematosus

While the immune reset induced by CD19 CAR T therapies has shown striking promise in systemic lupus erythematosus (SLE), the nature and duration of benefit mediated by B cell depletion remains unclear. Loss of tolerance in SLE is driven by extrafollicular and germinal center interactions between dendritic cells and self-reactive T and B cells. We hypothesized that CD19 CAR-engineered regulatory T cells (CAR19 EngTreg), capable of suppressing multiple autoimmune effector cell types, might promote long-term tolerance without producing high-levels of inflammatory mediators. To generate CAR19 EngTreg, we combined CD19 CAR delivery with homology directed repair at the FOXP3 locus to drive constitutive, stable FOXP3 expression in CD4 T cells. CAR19 EngTreg expressed canonical Treg markers; exhibited minimal inflammatory cytokine production; and blocked human B cell differentiation into antibody secreting plasma cells. In humanized mouse models, transfer of CAR19 EngTreg led to a reduction in B cell numbers with low inflammatory cytokine production relative to CD19 CAR Teff, and reduced tissue inflammation. In a short-term, in vivo imiquimod-induced inflammatory model, murine CAR19 EngTreg reduced skin pathology, spleen size, and inflammatory cytokines. Finally, in two distinct mouse models of lupus, CAR19 EngTreg persisted for >4 months leading to reduced autoimmune T and B effector populations, sustained reduction in multiple autoantibodies and improved renal function and histopathology. Thus, CAR19 EngTreg may offer a promising and durable therapeutic avenue for B cell-mediated autoimmune disorders capable of long-term regulation of both autoreactive B and T cells.

immunology↗

bioRxiv2024

Rigor and Reproducibility of Digital Spatial Profiling on Clinically Sourced Human Tissues

Spatial transcriptomic profiling enables precise quantification of gene expression with simultaneous localization of expression profiles onto tissue structures. This new technology promises to improve our understanding of the disease mechanisms. Therefore, there is intense interest in applying these methods in clinical trials or as laboratory developed tests to aid in diagnosis of disease. Before these technologies can be more broadly deployed in clinical research and diagnostics, it is necessary to thoroughly understand their performance in real world conditions. In this study, we vet technical reproducibility, data normalization methods and assay sensitivity focusing predominantly on one widely used spatial transcriptomic methodology, digital spatial profiling. Using clinically sourced human tissue specimens, we find that digital spatial profiling exhibits high rigor and reproducibility. Our approach lays the foundation for incorporation of digital spatial profiling methods into clinical workflows.

pathology↗

bioRxiv2023

The chromatin landscape of healthy and injured cell types in the human kidney

There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. However, comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measured dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We established a comprehensive and spatially-anchored epigenomic atlas to define the kidneys active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we noted distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3, KLF6, and KLF10 regulated the transition between health and injury, while in thick ascending limb cells this transition was regulated by NR2F1. Further, combined perturbation of ELF3, KLF6, and KLF10 distinguished two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.

molecular biology↗

bioRxiv2023

Programmable peroxidase-assisted signal amplification enables flexible detection of nucleic acid targets in cellular and histopathological specimens

In situ hybridization (ISH) is a powerful tool for investigating the spatial arrangement of nucleic acid targets in fixed samples. ISH is typically visualized using fluorophores to allow high sensitivity and multiplexing or with colorimetric labels to facilitate co-visualization with histopathological stains. Both approaches benefit from signal amplification, which makes target detection effective, rapid, and compatible with a broad range of optical systems. Here, we introduce a unified technical platform, termed pSABER, for the amplification of ISH signals in cell and tissue systems. pSABER decorates the in situ target with concatemeric binding sites for a horseradish peroxidase-conjugated oligonucleotide which can then catalyze the massive localized deposition of fluorescent or colorimetric substrates. We demonstrate that pSABER effectively labels DNA and RNA targets, works robustly in cultured cells and challenging formalin fixed paraffin embedded (FFPE) specimens. Furthermore, pSABER can achieve 25-fold signal amplification over conventional signal amplification by exchange reaction (SABER) and can be serially multiplexed using solution exchange. Therefore, by linking nucleic acid detection to robust signal amplification capable of diverse readouts, pSABER will have broad utility in research and clinical settings.

genomics↗

bioRxiv2022

Multi-omic Analysis of Primary Human Kidney Tissues Identifies Medulla-Specific Gene Expression Patterns

The renal medulla is a specialized region of the kidney with important homeostatic functions. It has also been implicated in genetic and developmental disorders and ischemic and drug-induced injuries. Despite its role in kidney function and disease, the medullas baseline gene expression and epigenomic signatures have not been well described in the adult human kidney. Here we generate and analyze gene expression (RNA-seq), chromatin accessibility (ATAC-seq) and chromatin conformation (Hi-C) data from adult human kidney cortex and medulla. Using data from our carefully annotated specimens, we assign samples in the larger public GTEx database to cortex and medulla, thereby identifying several misassignments and extracting meaningful medullary gene expression signatures. Using integrated analysis of gene expression, chromatin accessibility and conformation profiles, we reveal insights into medulla development and function. Our datasets will also provide a valuable resource for researchers in the GWAS community for functional annotation of genetic variants.

genetics↗

bioRxiv2021

Digital Spatial Profiling of Collapsing Glomerulopathy

Collapsing glomerulopathy is a histologically distinct variant of focal and segmental glomerulosclerosis that presents with heavy proteinuria and portends a poor prognosis. Collapsing glomerulopathy can be triggered by viral infections such as HIV and SARS-CoV-2. Transcriptional profiling of collapsing glomerulopathy lesions is difficult since only a few glomeruli may exhibit this histology within a kidney biopsy and the mechanisms driving this heterogeneity are unknown. Therefore, we used recently developed digital spatial profiling (DSP) technology which permits quantification of mRNA at the level of individual glomeruli. Using DSP, we profiled 1,852 transcripts in glomeruli from HIV and SARS-CoV-2 infected patients with biopsy confirmed collapsing glomerulopathy. The increased resolution of DSP uncovered heterogeneity in glomerular transcriptional profiles that were missed in early laser capture microdissection studies of pooled glomeruli. Focused validation using immunohistochemistry and RNA in situ hybridization showed good concordance with DSP results. Therefore, DSP represents a powerful method to dissect transcriptional programs of pathologically discernible kidney lesions.

pathology↗

bioRxiv2021

Multi-omic Characterization of Human Tubular Epithelial Cell Response to Serum

Proteinuria, the spillage of serum proteins into the urine, is a feature of glomerulonephritides, podocyte disorders and diabetic nephropathy. However, the response of tubular epithelial cells to serum protein exposure has not been systematically characterized. Using transcriptomic profiling we studied serum-induced changes in primary human tubular epithelial cells cultured in 3D microphysiological devices. Serum proteins induced cellular proliferation, cytokine secretion and activated a coordinated stress response. We orthogonally confirmed our findings by comparing the transcriptomic and epigenomic landscapes of intact human kidney cortex and isolated tubular epithelial cells cultured in fetal bovine serum. Importantly, key transcriptomic programs in response to either type of serum exposure remained consistent, including comparisons to an established mouse model of kidney injury. This serum-induced transcriptional response was dominated by switching off of nuclear receptor-driven programs and activation of AP-1 and NF-{kappa}B signatures in the tubular epigenomic landscape. These features of active regulation were seen at canonical kidney injury genes (HAVCR1) and genes associated with COVID-19 (ACE2, IL6). Our data provide a reference map for dissecting the regulatory and transcriptional response of kidney tubular epithelial cells injury induced by serum.

cell biology↗

bioRxiv2020

PaintSHOP enables the interactive design of transcriptome- and genome-scale oligonucleotide FISH experiments

Fluorescent in situ hybridization (FISH) allows researchers to visualize the spatial position and quantity of nucleic acids in fixed samples. Recently, considerable progress has been made in developing oligonucleotide (oligo)-based FISH methods. These methods have enabled researchers to study the three-dimensional organization of the genome at super-resolution and visualize the spatial patterns of gene expression for thousands of genes in individual cells. While considerable progress has been made in developing new molecular methods that harness complex oligo libraries for FISH, there are few existing computational tools to support the bioinformatics workflows necessary to carry out these experiments. Here, we introduce Paint Server and Homology Optimization Pipeline (PaintSHOP), an interactive platform for the reproducible design of oligo FISH experiments. PaintSHOP enables researchers to identify probes for their experimental targets efficiently, to incorporate additional necessary sequences such as primer pairs, and to easily generate standardized files documenting the design of their libraries. Our platform integrates a machine learning model that quantitatively predicts probe specificity on the genome scale into a dynamic web application that creates ready-to-order probe sets for a wide variety of applications. The goal of this freely available web resource is to democratize and standardize the process of designing complex probe sets for the oligo FISH community. PaintSHOP can be accessed at: paintshop.ioCompeting Interest StatementThe authors have declared no competing interest.View Full Text

genomics↗

bioRxiv2018

Cryptic Promoter Activation Drives POU5F1 (OCT4) Expression in Renal Cell Carcinoma

Transcriptional dysregulation drives cancer formation but the underlying mechanisms are still poorly understood. As a model system, we used renal cell carcinoma (RCC), the most common malignant kidney tumor which canonically activates the hypoxia-inducible transcription factor (HIF) pathway. We performed genome-wide chromatin accessibility and transcriptome profiling on paired tumor/normal samples and found that numerous transcription factors with a RCC-selective expression pattern also demonstrated evidence of HIF binding in the vicinity of their gene body. Some of these transcription factors influenced the tumors regulatory landscape, notably the stem cell transcription factor POU5F1 (OCT4). Unexpectedly, we discovered a HIF-pathway-responsive cryptic promoter embedded within a human-specific retroviral repeat element that drives POU5F1 expression in RCC via a novel transcript. Elevat POU5F1 expression levels were correlated with advanced tumor stage and poorer overall survival in RCC patients. Thus, integrated transcriptomic and epigenomic analysis of even a small number of primary patient samples revealed remarkably convergent shared regulatory landscapes and a novel mechanism for dysregulated expression of POU5F1 in RCC.

cancer biology↗