Biology subjects

Greka, A.

Publications and source records attributed to Greka, A..

15 recordsLinked to original sources

bioRxiv2026

An Integrated Atlas of the Human Kidney Spanning Health and Diseases

The human kidney contains highly specialized cell populations. Despite numerous single-cell and single-nucleus transcriptomics studies, differences in cohorts, technologies, analytical pipelines, and annotation frameworks have limited the ability to define consensus kidney cell states, identify disease-associated populations and interpret kidney disease genetic susceptibility. Here, we assembled 18 human kidney single-cell and single-nucleus RNA-sequencing datasets spanning 232 donors and five major disease contexts into a uniformly processed and computationally integrated Human Kidney Cell Atlas (HKCA), comprising over one million high-quality cells (816,895) and nuclei (215,308). The HKCA resolves 63 cell types and 120 harmonized cell states, including rare epithelial and stromal populations associated with kidney diseases . Integration with spatial transcriptomics, intercellular communication networks, and human genetic association data further defined the anatomical context and disease relevance of these populations. The HKCA also provides a framework for automated annotation of independent kidney human and mouse datasets. Together, the HKCA establishes a comprehensive reference for human kidney biology, enabling disease interpretation and genetic risk localization at cellular resolution.

genomics↗

bioRxiv2026

Double-bond geometry determines fatty acid metabolic fate and ferroptosis sensitivity

Ferroptosis is driven by the accumulation of oxidatively damaged membrane phospholipids, making membrane lipid composition a central determinant of cell death sensitivity. While fatty acid chain length and degree of unsaturation are well-established regulators of ferroptosis, whether fatty acid stereochemistry contributes to ferroptosis susceptibility is mostly unexplored. Here, we systematically screened structurally diverse fatty acids for their ability to modulate ferroptosis and unexpectedly identified trans-unsaturated fatty acids as potent sensitizers. Compared with its cis counterpart linoleic acid, the trans polyunsaturated fatty acid (PUFA) linoelaidic acid more strongly enhanced lipid peroxidation and promoted the accumulation of ferroptosis-susceptible phospholipid species. Unexpectedly, the trans monounsaturated fatty acid petroselaidic acid also sensitized cells to ferroptosis, whereas its cis stereoisomer petroselinic acid suppressed ferroptosis. Mechanistically, petroselaidic acid required stearoyl-CoA desaturase-dependent conversion to a PUFA, directly demonstrating that double-bond geometry can redirect fatty acid metabolic fate through altered recognition by lipid metabolic enzymes. Although linoelaidic acid and petroselaidic acid followed distinct metabolic pathways, both converged on phospholipid remodeling that expanded pools of ferroptosis-susceptible membrane lipids. Together, our findings demonstrate that fatty acid double-bond geometry determines their metabolic fate and the membrane phospholipid composition, establishing lipid stereochemistry as a previously unrecognized structural determinant of ferroptosis sensitivity.

cell biology↗

bioRxiv2024

FAF2 is a bifunctional regulator of peroxisomal homeostasis and saturated lipid responses

Exposure to saturated fatty acids (SFAs), such as palmitic acid, can lead to cellular metabolic dysfunction known as lipotoxicity. Although canonical adaptive metabolic processes like lipid storage or desaturation are known cellular responses to saturated fat exposure, the link between SFA metabolism and organellar biology remains an area of active inquiry. We performed a genome-wide CRISPR knockout screen in human epithelial cells to identify modulators of SFA toxicity. The screen revealed peroxisomal proteins, especially those that impact ether lipid synthesis, as important regulators of lipotoxicity. We identified Fas-associated factor family member 2 (FAF2) as a critical bifunctional co-regulator of peroxisomal and fatty acid biology. We further uncovered a new biological function for the ubiquitin-regulatory X (UBX) and UAS thioredoxin-like domains of FAF2, demonstrating their requirement for peroxisomal protein abundance and SFA-induced cellular stress. Our work highlights the role of FAF2 in regulating peroxisomal abundance and function, and the peroxisome as a key organelle in the cellular response to SFAs.

cell biology↗

bioRxiv2023

FALCON systematically interrogates free fatty acid biology and identifies a novel mediator of lipotoxicity

Cellular exposure to free fatty acids (FFA) is implicated in the pathogenesis of obesity-associated diseases. However, studies to date have assumed that a few select FFAs are representative of broad structural categories, and there are no scalable approaches to comprehensively assess the biological processes induced by exposure to diverse FFAs circulating in human plasma. Furthermore, assessing how these FFA- mediated processes interact with genetic risk for disease remains elusive. Here we report the design and implementation of FALCON (Fatty Acid Library for Comprehensive ONtologies) as an unbiased, scalable and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids (MUFAs) with a distinct lipidomic profile associated with decreased membrane fluidity. Furthermore, we developed a new approach to prioritize genes that reflect the combined effects of exposure to harmful FFAs and genetic risk for type 2 diabetes (T2D). Importantly, we found that c-MAF inducing protein (CMIP) protects cells from exposure to FFAs by modulating Akt signaling and we validated the role of CMIP in human pancreatic beta cells. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism. HighlightsO_LIFALCON (Fatty Acid Library for Comprehensive ONtologies) enables multimodal profiling of 61 free fatty acids (FFAs) to reveal 5 FFA clusters with distinct biological effects C_LIO_LIFALCON is applicable to many and diverse cell types C_LIO_LIA subset of monounsaturated FAs (MUFAs) equally or more toxic than canonical lipotoxic saturated FAs (SFAs) leads to decreased membrane fluidity C_LIO_LINew approach prioritizes genes that represent the combined effects of environmental (FFA) exposure and genetic risk for disease C_LIO_LIC-Maf inducing protein (CMIP) is identified as a suppressor of FFA-induced lipotoxicity via Akt-mediated signaling C_LI

cell biology↗

bioRxiv2022

Accelerated cognitive decline in obese mouse model of Alzheimer's disease is linked to sialic acid-driven immune deregulation

Systemic immunity supports healthy brain homeostasis. Accordingly, conditions causing systemic immune deregulation may accelerate onset of neurodegeneration in predisposed individuals. Here we show that, in the 5xFAD mouse model of Alzheimers disease (AD), high-fat diet-induced obesity accelerated cognitive decline, which was associated with immune deviations comprising increased splenic frequencies of exhausted CD4+ T effector memory cells and CD4+FOXP3+ regulatory T cells (Tregs). Non-targeted plasma metabolomics identified N-acetylneuraminic acid (NANA), the predominant sialic acid, as the major obesity-induced metabolite in 5xFAD mice, the levels of which directly correlated with Tregs abundance and inversely correlated with cognitive performance. Visceral adipose tissue macrophages were identified by sNuc-Seq as one potential source of NANA. Exposure to NANA led to immune deregulation in middle-aged wild-type mice, and ex vivo in human T cells. Our study identified diet-induced immune deregulation, potentially via sialic acid, as a previously unrecognized link between obesity and AD.

neuroscience↗

bioRxiv2021

High Resolution Slide-seqV2 Spatial Transcriptomics Enables Discovery of Disease-Specific Cell Neighborhoods and Pathways

High resolution spatial transcriptomics is a transformative technology that enables mapping of RNA expression directly from intact tissue sections; however, its utility for the elucidation of disease processes and therapeutically actionable pathways remain largely unexplored. Here we applied Slide-seqV2 to mouse and human kidneys, in healthy and in distinct disease paradigms. First, we established the feasibility of Slide-seqV2 in human kidney by analyzing tissue from 9 distinct donors, which revealed a cell neighborhood centered around a population of LYVE1+ macrophages. Second, in a mouse model of diabetic kidney disease, we detected changes in the cellular organization of the spatially-restricted kidney filter and blood flow regulating apparatus. Third, in a mouse model of a toxic proteinopathy, we identified previously unknown, disease-specific cell neighborhoods centered around macrophages. In a spatially-restricted subpopulation of epithelial cells, we also found perturbations in 77 genes associated with the unfolded protein response (UPR). Our studies illustrate and experimentally validate the utility of Slide-seqV2 for the discovery of disease-specific cell neighborhoods. One-Sentence SummaryHigh resolution Slide-seqV2 spatial transcriptomics in human and mouse kidneys.

molecular biology↗

bioRxiv2021

Single-nucleus cross-tissue molecular reference maps to decipher disease gene function

Understanding the function of genes and their regulation in tissue homeostasis and disease requires knowing the cellular context in which genes are expressed in tissues across the body. Single cell genomics allows the generation of detailed cellular atlases in human tissues, but most efforts are focused on single tissue types. Here, we establish a framework for profiling multiple tissues across the human body at single-cell resolution using single nucleus RNA-Seq (snRNA-seq), and apply it to 8 diverse, archived, frozen tissue types (three donors per tissue). We apply four snRNA-seq methods to each of 25 samples from 16 donors, generating a cross-tissue atlas of 209,126 nuclei profiles, and benchmark them vs. scRNA-seq of comparable fresh tissues. We use a conditional variational autoencoder (cVAE) to integrate an atlas across tissues, donors, and laboratory methods. We highlight shared and tissue-specific features of tissue-resident immune cells, identifying tissue-restricted and non-restricted resident myeloid populations. These include a cross-tissue conserved dichotomy between LYVE1- and HLA class II-expressing macrophages, and the broad presence of LAM-like macrophages across healthy tissues that is also observed in disease. For rare, monogenic muscle diseases, we identify cell types that likely underlie the neuromuscular, metabolic, and immune components of these diseases, and biological processes involved in their pathology. For common complex diseases and traits analyzed by GWAS, we identify the cell types and gene modules that potentially underlie disease mechanisms. The experimental and analytical frameworks we describe will enable the generation of large-scale studies of how cellular and molecular processes vary across individuals and populations.

cell biology↗

bioRxiv2021

Obesity-instructed TREM2high macrophages identified by comparative analysis of diabetic mouse and human kidney at single cell resolution

Mouse models are a tool for studying the mechanisms underlying complex diseases; however, differences between species pose a significant challenge for translating findings to patients. Here, we used single-cell transcriptomics and orthogonal validation approaches to provide cross-species taxonomies, identifying shared broad cell classes and unique granular cellular states, between mouse and human kidney. We generated cell atlases of the diabetic and obese kidney using two different mouse models, a high-fat diet (HFD) model and a genetic model (BTBR ob/ob), at multiple time points along disease progression. Importantly, we identified a previously unrecognized, expanding Trem2high macrophage population in kidneys of HFD mice that matched human TREM2high macrophages in obese patients. Taken together, our cross-species comparison highlights shared immune and metabolic cell-state changes.

genomics↗

bioRxiv2021

PIP2 regulation of TRPC5 channel activation and desensitization

Transient receptor potential canonical type 5 (TRPC5) channels are expressed in the brain and kidney, and have been identified as promising therapeutic targets whose selective inhibition can protect against diseases driven by a leaky kidney filter. They are activated by elevated levels of extracellular Ca2+ or application of lanthanide ions but also by G protein (Gq/11) stimulation. Phosphatidylinositol bis-phosphate (PIP2) hydrolysis leads to protein kinase C- (PKC-) mediated phosphorylation of TRPC5 channels and desensitization of their activity. Even though PIP2 regulation of TRP channels is being widely studied, the roles of PIP2 in maintaining TRPC5 channel activity, the PIP2 involvement in channel stimulation by its hydrolysis product diacyl glycerol (DAG), or the desensitization of activity by DAG-stimulated PKC activity remain unclear. Here, we show that PIP2 controls both the PKC-mediated inhibition of TRPC5 currents as well as the activation by DAG and lanthanides and that it accomplishes this through control of gating rather than channel cell surface density. The mechanistic insights achieved by the present work promise to aid in the development of more selective and precise molecules to block TRPC5 channel activity and illuminate new therapeutic opportunities for targeted therapies for a group of diseases for which there is currently a great unmet need.

physiology↗

bioRxiv2020

Human relevance of blocking the Rac1-TRPC5 pathway as a podocyte-protective strategy for progressive kidney diseases

Podocyte injury and the appearance of proteinuria are key features of several progressive kidney diseases. Genetic deletion or selective inhibition of TRPC5 channels with small-molecule inhibitors protects podocytes in rodent models of disease, but less is known about the human relevance and translatability of TRPC5 inhibition. Here, we investigate the effect of TRPC5 inhibition in puromycin aminonucleoside (PAN)-treated human iPSC-derived podocytes and kidney organoids. We first established that systemic administration of the TRPC5-specific blocker AC1903 was sufficient to protect podocyte cytoskeletal proteins and suppress proteinuria in PAN-induced nephrosis in rats, an established model of podocyte injury and progressive kidney disease. PAN treatment also triggered the Rac1-TRPC5 injury pathway in human iPSC-derived podocytes and kidney organoids. TRPC5 current was recorded in human iPSC-derived podocytes, and was blocked by AC1903. The TRPC5 blocker also reversed the effects of PAN-induced injury in human podocytes in both 2D and 3D culture systems. Taken together, these results revealed the relevance of the TRPC5-Rac1 pathway in human kidney tissue highlighting the potential of this therapeutic strategy for patients.

cell biology↗

bioRxiv2020

Single cell transcriptomics reveal disrupted kidney filter cell-cell interactions after early and selective podocyte injury

The health of the kidney filtration barrier requires communication between podocytes, endothelial cells and mesangial cells. Disruption of these cell-cell interactions is thought to contribute to disease progression in chronic kidney diseases (CKD). We recently demonstrated that podocyte ablation via doxycycline-inducible deletion of an essential endogenous molecule, CTCF (iCTCFpod-/-), is sufficient to drive progressive CKD. However, the earliest events connecting podocyte injury to disrupted intercellular communication within the kidney filter remain unclear. Here we performed single-cell RNA sequencing of kidney tissue from iCTCFpod-/- mice after one week of doxycycline induction to generate a map of the earliest transcriptional effects of podocyte injury on cell-cell interactions at single cell resolution. A subset of podocytes showed the earliest signs of injury due to disrupted gene programs for cytoskeletal regulation and mitochondrial function. Surviving podocytes upregulated Col4a5, causing reactive changes in integrin expression in endothelial populations and mesangial cells. Intercellular interaction analysis revealed several receptor-ligand-target gene programs as drivers of endothelial cell injury and abnormal matrix deposition. This analysis reveals the earliest disruptive changes within the kidney filter, pointing to new, actionable targets within a therapeutic window that may allow us to maximize the success of much needed therapeutic interventions for CKD.

cell biology↗

bioRxiv2020

A High Content Screen for Mucin-1-Reducing Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury during the COVID-19 pandemic

Drug repurposing is the only method capable of delivering treatments on the shortened time-scale required for patients afflicted with lung disease arising from SARS-CoV-2 infection. Mucin-1 (MUC1), a membrane-bound molecule expressed on the apical surfaces of most mucosal epithelial cells, is a biochemical marker whose elevated levels predict the development of acute lung injury (ALI) and respiratory distress syndrome (ARDS), and correlate with poor clinical outcomes. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce MUC1 protein abundance. Our screen identified Fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, Fostamatinib reduced MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by Fostamatinib promoted MUC1 removal from the cell surface. Our work reveals Fostamatinib as a repurposing drug candidate for ALI and provides the rationale for rapidly standing up clinical trials to test Fostamatinib efficacy in patients with COVID-19 lung injury.

cell biology↗

bioRxiv2020

RAAS blockade, kidney disease, and expression of ACE2, the entry receptor for SARS-CoV-2, in kidney epithelial and endothelial cells

SARS-CoV-2, the coronavirus that causes COVID-19, binds to angiotensin-converting enzyme 2 (ACE2) on human cells. Beyond the lung, COVID-19 impacts diverse tissues including the kidney. ACE2 is a key member of the Renin-Angiotensin-Aldosterone System (RAAS) which regulates blood pressure, largely through its effects on the kidney. RAAS blockers such as ACE inhibitors (ACEi) and Angiotensin Receptor Blockers (ARBs) are widely used therapies for hypertension, cardiovascular and chronic kidney diseases, and therefore, there is intense interest in their effect on ACE2 expression and its implications for SARS-CoV-2 pathogenicity. Here, we analyzed single-cell and single-nucleus RNA-seq of human kidney to interrogate the association of ACEi/ARB use with ACE2 expression in specific cell types. First, we performed an integrated analysis aggregating 176,421 cells across 49 donors, 8 studies and 8 centers, and adjusting for sex, age, donor and center effects, to assess the relationship of ACE2 with age and sex at baseline. We observed a statistically significant increase in ACE2 expression in tubular epithelial cells of the thin loop of Henle (tLoH) in males relative to females at younger ages, the trend reversing, and losing significance with older ages. ACE2 expression in tLoH increases with age in females, with an opposite, weak effect in males. In an independent cohort, we detected a statistically significant increase in ACE2 expression with ACEi/ARB use in epithelial cells of the proximal tubule and thick ascending limb, and endothelial cells, but the association was confounded in this small cohort by the underlying disease. Our study illuminates the dynamics of ACE2 expression in specific kidney cells, with implications for SARS-CoV-2 entry and pathogenicity.

genomics↗

bioRxiv2020

HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes

Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of up to 100 chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome scRNA-seq, making HyPR-seq a powerful method for targeted RNA profiling in single cells.

genomics↗

bioRxiv2019

Kidney organoid reproducibility across multiple human iPSC lines and diminished off target cells after transplantation revealed by single cell transcriptomics

Human iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we used single cell RNA-Seq (scRNA-Seq) to profile 415,775 cells to show that organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes were largely reproducible across iPSC lines, time points, protocols, and replicates, cell proportions were variable between different iPSC lines. Off-target cell proportions were the most variable. Prolonged in vitro culture did not alter cell types, but organoid transplantation under the mouse kidney capsule diminished off-target cells. Our work shows how scRNA-seq can help score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells.

genomics↗